UNIVERSITY  OF  CALIFORNIA 
SAN  FRANCISCO  LIBRARY 


IMMUNITY 

IN 

INFECTIVE  DISEASES 


BY 
METCHNIKOFF 

FOREIGN  MEMBER  OF  THE  ROYAL   SOCIETY  OF  LONDON 
PROFESSOR  AT  THE  PASTEUR  INSTITUTE,   PARIS 


TRANSLATED  FROM  THE  FRENCH 

BY 

FRANCIS   G.   BINNIE 

OF  THE  PATHOLOGICAL  DEPARTMENT,  UNIVERSITY  OF  CAMBRIDGE 


CAMBRIDGE 

AT  THE  UNIVERSITY  PRESS 
1907 


First  Edition  1905, 
Reprinted    1907 


PREFACE  TO  THE  ENGLISH  EDITION. 

TN   preparing   for   the    English-reading   student    this  version    of 

M.  Metchnikoff's  latest  work,  wherein  he  "sums  up  the  labours 
of  twenty-five  years,"  it  has  been  my  aim  to  give  a  faithful  rendering 
of  the  ideas  and  argument  of  the  original,  even  at  the  risk  of  an 
occasional  crude  expression,  rather  than  to  attempt  to  reproduce 
the  brilliancy  of  the  original  by  any  wide  verbal  departure  from 
the  text. 

The  Table  of  Contents  forms  an  admirable  analytical  summary  of 
the  main  subject-matters  treated,  but  an  alphabetical  Index  has  been 
added  to  the  present  edition,  and,  though  not  at  all  exhaustive,  this 
may  serve  as  a  key  to  the  many  authors  cited  and  to  the  maze  of 
detail  discussed  in  the  work. 

The  marginal  reference  to  the  pages  of  the  original  work  will, 
I  hope,  commend  itself  to  those  readers  who  may  wish  to  refer  to 
the  ipsissima  verba  of  the  author.  It  is,  I  believe,  a  novelty  in 
scientific  works,  though  familiar  in  works  in  other  departments  of 
literature. 

I  am  under  deep  obligations  to  Professor  Woodhead  (who  has 
read  the  whole  of  the  proofs)  and  to  Mr  A.  E.  Shipley,  and 
Mr  G.  H.  F.  Nuttall  (who  have  read  portions)  for  much  valuable 
criticism  and  advice. 

THE  TRANSLATOR 

August,  1905. 


TO  MESSIEURS  E.  DUCLAUX  AND  E.  ROUX. 


My  dear  Friends, 

Permit  me  to  dedicate  to  you  this  work,  which  sums  up 
the  labours  of  twenty-five  years ;  a  very  great  part  of  it  has  been 
carried  out  by  your  side,  you  who  have  done  so  much  to  lighten 
my  task. 

When,  nearly  fourteen  years  ago,  you  allowed  me  to  share  your 
work  alongside  the  venerated  Master  who  founded  the  House  where 
we  have  laboured  together,  you  were  anything  but  partisans  of 
my  theories;  they  seemed  to  you  too  vitalistic,  and  not  sufficiently 
physico-chemical.  In  course  of  time  you  became  convinced  that 
my  ideas  were  not  without  foundation,  and  since  then  you  have 
given  me  warm  encouragement  to  pursue  my  researches  in  the 
field  that  I  had  marked  out  for  myself. 

Working  by  your  side  and  drawing  largely  from  your  vast 
and  varied  stores  of  knowledge,  I  felt  myself  safe  from  those  diva- 
gations into  which  a  zoologist,  who  had  wandered  into  the  domain 
of  biological  chemistry  and  of  medical  science,  is  likely  to  stray. 
I  tliank  you  with  aU  my  heart,  and  I  beg  you  to  accept  the  homage 
of  this  work  as  a  testimony  of  my  deepest  gratitude  and  of  my 
warmest  friendship. 


ELIE  METCHOTKOFF. 


Institut  Pasteur, 

3  October,  1901. 


PREFACE. 


WHEN",  ten  years  ago,  I  was  preparing  my  Lessons  on  the  Compa- 
rative Pathology  of  Inflammation  for  the  press,  I  hoped  that 
the  other  sections  of  the  phagocytic  theory — Immunity,  Atrophies, 
and  Healing — would  soon  follow  this  first  work.  This  hope  has  not 
been  realised,  and  it  has  needed  prolonged  work  ere  I  could  publish 
the  volume  I  have  just  completed. 

During  this  long  period  I  sent  out  several  ballons  d'essai  under 
the  form  of  summaries  of  the  question  of  Immunity,  published  in  the 
Semaine  medicale  (1892),  the  Ergebnisse  of  Lubarsch  and  Ostertag 
(1886),  and  the  Handbuch  der  Hygiene  by  Weyl  (1897).  I  there 
attempted,  as  far  as  possible,  to  give  a  general  picture  of  the 
phenomena  of  Immunity  in  the  infective  diseases,  and  it  was  my 
desire  to  excite  criticism  and  opposition,  in  order  to  determine  the 
fate  of  the  theory  of  phagocytes  in  its  application  to  the  problem  of 
Immunity. 

The  most  recent  attempt  in  this  direction  was  made  at  the  Inter- 
national Congress  at  Paris,  in  the  past  year  (1900),  when  I  presented 
my  report  on  Immunity  before  an  audience  which  included,  amongst 
others,  my  principal  opponents.  It  was  the  result  of  this  Congress 
which  at  length  decided  me  to  bring  together  my  vie^ •*  on  Immunity 
in  a  volume  which  I  now  present  to  the  reader. 

Convinced  that  many  of  the  objections  raised  against  the  phago- 
cytic theory  of  Immunity  proceeded  solely  from  an  insufficient 
knowledge  of  the  theory,  I  thought  that  a  work  condensed  into  one 
volume  might  render  some  service  to  those  who  are  interested  in 
the  problem  of  Immunity.  I  do  not  know  whether  I  shall  convert  my 
opponents,  but  I  am  convinced  that  a  perusal  of  this  book  will  clear 
away  certain  misunderstandings.  A  very  competent  observer  recently 
confessed  in  one  of  his  publications  that  for  many  years  he  had 
been  unaware  of  the  experiments  of  M.  J.  Bordet  and  myself  on 
Immunity  against  the  cholera  vibrio,  experiments  which  he  now 


x  Preface 

regards  as  of  fundamental  importance  for  the  comprehension  of  the 
whole  question  of  Immunity.  I  hope  that  after  the  appearance  of 
this  treatise  such  oversights  will  not  be  so  likely  to  occur. 

Should  I  not  succeed  in  convincing  my  opponents  of  the  justice 
of  the  cause  which  I  defend,  I  shall  at  least  have  informed  my  critics 
and  shall  have  given  them  an  opportunity  of  discussing  it  with  a 
thorough  knowledge  of  the  material  on  which  it  is  based.  This 
result  alone  would  justify  me  in  having  undertaken  this  work. 

At  first  I  intended  to  add  to  my  explanation  of  Immunity  a 
theory  of  the  phenomena  of  healing  in  infective  diseases,  but  I  soon 
had  to  renounce  this  project,  for  its  execution  would  have  increased 
too  greatly  the  bulk  of  the  book  which,  without  it,  has  already  assumed 
considerable  proportions.  It  seemed  to  me  preferable  to  set  forth 
the  present  state  of  the  question  without  paying  too  much  attention 
to  the  historical  sequence  of  the  discoveries,  and  to  reserve  for  a 
special  chapter,  at  the  end  of  the  work,  a  sketch  of  the  history  of  our 
knowledge  on  Immunity. 

Before  I  ask  the  reader  to  glance  through  this  work,  I  should 
mention  that  I  have  been  heartily  seconded  in  its  preparation  by  many 
of  my  friends  and  collaborators.  I  offer  my  most  sincere  thanks  to 
MM.  Roux,  Nocard,  Massart,  and  J.  Bordet,  who  kindly  undertook 
to  read  my  manuscript  throughout,  or  such  parts  of  it  as  related  to 
their  special  subjects.  For  example,  M.  Nocard  rendered  me  a 
very  great  service  by  correcting  the  paragraphs  of  Chapter  xv, 
which  treat  of  the  vaccinations  against  epizootic  diseases,  and 
M.  Massart,  by  giving  me  his  advice  on  the  subject  of  immunity 
in  plants. 

I  owe  very  special  thanks  to  M.  Mesnil,  who  has  been  good 
enough  to  give  me  very  effective  help  in  the  dry  task  of  correcting 
the  manuscript  and  proofs. 

I  beg  MM.  E.  Re"my  and  L.  Barneoud  to  accept  my  thanks  for  the 
care  they  have  bestowed  on  the  execution  of  the  illustrations  in  this 
work. 


METCHNIKOFF. 


PARIS,  INSTITUT  PASTEUR, 
3  October,  1901. 


CONTENTS. 

PAGE 

PREFACE  TO  THE  ENGLISH  EDITION v 

PREFACE ix 

INTRODUCTION      ........        1 

Importance  of  the  study  of  immunity  from  a  general  point  of  view. — Part 
played  by  parasites  in  infective  diseases. — Intoxications  by  the  products 
of  micro-organisms. — Resistance  of  the  organism  to  the  invasion  of  micro- 
organisms. 

Xatural  immunity  and  acquired  immunity. 

Immunity  to  micro-organisms  and  immunity  to  toxins. 

CHAPTER   I 
IMMUNITY  IN  UNICELLULAR  ORGANISMS 11 

Infective  diseases  of  the  unicellular  organisms. — Intracellular  digestion  in 
the  Protozoa. — Ainoebo-diastase. — Part  played  by  digestion  in  the 
defence  of  the  Protozoa  against  infective  parasites. — Defences  of  the 
Paramaecia  against  micro-organisms. — Part  played  by  irritability  in 
defence  in  the  lower  organisms. 

Immunity  of  unicellular  organisms  to  toxins. — Acclimatisation  of  bacteria 
to  toxic  substances. — Protective  secretion  of  membranes  by  bacteria. 

Adaptation  of  the  Protozoa  to  saline  solutions — of  yeasts  to  poisons — of 
yeasts  to  milk-sugar. 

Irritability  of  unicellular  organisms  and  Weber- Fechner's  psycho-physical  law. 

CHAPTER   II 

IMMUNITY  IN  MULTICELLULAR  PLANTS 29 

Infective  diseases  of  plants.— Plasmodia  of  the  Myxomycetes  and  their 
chemiotaxis. — Adaptation  of  the  plasmodia  to  poisons.  —  Pathogenic 
action  of  Sclerotinia  upon  Phanerogams. — The  cicatrisation  of  plants. — 
Defence  in  plants  against  Bacteria. — Sensitiveness  of  vegetable  cells  to 
osmotic  pressure. — Adaptation  of  plants  to  modifications  of  osmotic 
pressure. — Dependence  of  the  chemical  phenomena  upon  the  irritability 
of  the  vegetable  cells.— The  law  of  Weber-Fechner. 

CHAPTER   III 

PRELIMINARY  REMARKS  ox  IMMUNITY  IN  THE  ANIMAL  KINGDOM     ...       40 

Examples  of  natural  immunity  among  the  Invertebrates. — Immunity  against 
micro-organisms  and  insusceptibility  to  microbial  poisons  are  two 
distinct  properties. — The  refractory  organism  does  not  eliminate  micro- 
organisms by  the  excretory  channels^— It  destroys  them  by  a  process  of 


xii  Contents 

PAGE 

resorption.— The  fate  of  foreign  bodies  in  the  organism. — The  resorption 
of  cells. — Intracellular  digestion. — This  digestion  effected  by  the  aid  of 
soluble  ferments.  —  Digestion  in  Planarians  and  Actinians.  —  Actino- 
diastase.— Transition  from  intracellular  digestion  to  digestion  by  secreted 
juices. — Digestion  in  the  higher  animals. — Enterokynase  and  the  part 
it  plays  in  digestion. — The  psychical  and  nervous  elements  in  digestion. 
— Adaptation  of  the  pancreatic  secretion  to  the  kind  of  food.— Excretion 
of  pepsin  in  the  blood  and  in  the  urine. 

CHAPTER   IV 

RESORPTION  OF  THE  FORMED  ELEMENTS  67 

Digestion  in  the  tissues.— Resorption  of  cells  in  the  Invertebrata.— Resorption 
of  red  corpuscles  by  the  phagocytes  of  the  Vertebrata. — Phagocytes. — 
Various  categories  of  these  cells. — Macrophages  and  microphages. — Part 
played  by  macrophages  in  the  resorption  of  the  formed  elements. — 
Digestive  property  of  the  macrophagic  organs.— Solution  of  the  red 
blood  corpuscles  by  the  blood  serums.  —  The  two  substances  which 
operate  in  haemolysis.  Macrocytase  and  fixative. — Analogy  of  the  latter 
with  enterokynase.—  Escape  of  the  macrocytase  during  phagolysis.  Sup- 
pression of  phagolysis.  Resorption  of  the  spermatozoa. — Presence  of 
fixatives  in  plasmas.— Origin  of  fixatives. 

CHAPTER    V 
RESORPTION  OF  ALBUMINOID  FLUIDS 106 

Resorption  of  albuminoid  substances. — The  precipitins  of  blood  serum  which 
appear  as  a  result  of  the  absorption  of  serums  and  of  milk. — Absorption 
of  gelatine. — Leucocytic  origin  of  the  ferment  which  digests  gelatine. — 
Anti-enzymes.  —  Antirennet.  —  The  anticytotoxins.  —  Antihaemotoxic 
serums. —  Their  two  constituent  parts:  anticytase  and  antifixative. — 
Action  of  anticytase. — The  antispermotoxins. — Origin  of  anticytotoxins. — 
Ehrlich's  theory  on  this  question. — Origin  of  antihaemotoxin. — Origin  of 
antispermotoxin.  —  Production  of  this  antibody  by  castrated  males.  — 
The  antispermofixative  produced  when  the  spermatozoa  are  excluded. 
— Distribution  of  spermotoxin  and  antispermotoxin  in  the  organism. 

CHAPTER    VI 
NATURAL  IMMUNITY  AGAINST  PATHOGENIC  MICRO-ORGANISMS    ....      128 

Natural  immunity  and  the  composition  of  the  body  fluids. — Cultivation  of  the 
bacteria  of  influenza  and  pleuro-pneumonia  in  the  fluids  of  refractory 
animals. — Resistance  of  Daphniae  to  the  Blastomycetes. — Examples  of 
natural  immunity  in  Insects  and  Mollusca. — Immunity  of  Fishes  against 
the  anthrax  bacillus. — Immunity  of  frogs  against  anthrax,  Ernst's  bacillus, 
the  bacillus  of  mouse  septicaemia,  and  the  cholera  vibrio.  —  Natural 
immunity  in  the  cayman. — Immunity  of  the  fowl  and  pigeon  against 
anthrax  and  human  tuberculosis. — Immunity  of  the  dog  and  rat  against 
the  anthrax  bacillus. — Immunity  of  Mammals  against  anthrax  vaccines. — 
Immunity  of  the  guinea-pig  against  spirilla,  vibrios,  and  streptococci. — 
Natural  immunity  against  anaerobic  bacilli. — Fate  of  Blastomycetes  and 
Trypanosomata  in  the  refractory  organism. 


Contents  xiii 

CHAPTER    VH 

PAGE 
THE  MECHANISM    OF   NATURAL  IMMUNITY  AGAINST   MICRO-ORGANISMS  .  .         175 

The  destruction  of  micro-organisms  in  natural  immunity  is  an  act  of  re- 
sorption. — Part  played  by  inflammation  in  natural  immunity. — Importance 
of  microphages  in  immunity  against  micro-organisms. — Chemiotaxis  of 
leucocytes  and  ingestion  of  micro-organisms. — Phagocytes  are  capable 
of  ingesting  living  and  virulent  micro-organisms. — The  digestion  of  micro- 
organisms in  phagocytes  is  most  often  effected  in  a  feebly  acid  medium. — 
Bactericidal  property  of  serums. — Phagocytic  origin  of  the  bactericidal 
substance. — Theory  of  the  secretion  of  the  bactericidal  substance  by 
leucocytes. — Comparison  of  the  bactericidal  power  of  serums  and  of 
blood  plasmas. — The  bactericidal  substance  of  blood  serums  must  not 
be  considered  a  secretion-product  of  leucocytes ;  it  remains  within  the 
phagocytes,  so  long  as  they  are  intact. — The  cytases. — Two  kinds  of 
cytases  :  macrocytase  and  microcytase. — Cytases  are  endo-enzymes,  allied 
to  trypsins. — Changes  in  the  staining  properties  and  in  the  form  of  micro- 
organisms in  the  phagocytes. — Absence  or  rarity  of  fixatives  in  the 
serums  of  animals  endowed  with  natural  immunity. — The  agglutination 
of  micro-organisms  does  not  play  any  important  part  in  the  mechanism 
of  natural  immunity. — Absence  of  antitoxic  property  of  the  body  fluids 
in  natural  immunity.  —  The  phagocytes  destroy  the  micro-organisms 
without  their  ingestion  being  preceded  by  neutralisation  of  the  toxins. 

CHAPTER  YIII 

SURVEY  OF  THE  FACTS  BEARING  ON  ACQUIRED  IMMUNITY  AGAINST  MICRO- 
ORGANISMS   207 

The  discovery  of  attenuated  viruses  and  its  application  to  vaccination  against 
infective  diseases. — Vaccination  by  microbial  products. — Vaccination  with 
serums. — The  acquired  immunity  of  the  frog  against  pyocyanic  disease. 
— The  acquired  immunity  against  vibrios. — Extracellular  destruction  of 
the  cholera  vibrio. — Part  played  by  two  substances  in  Pfeiffer's  pheno- 
menon.— Specificity  of  fixatives. — Phagolysis  and  its  relation  to  the  extra- 
cellular destruction  of  vibrios.  —  Part  played  by  phagocytosis  in  the 
acquired  immunity  against  vibrios. — Fate  of  the  spirilla  of  recurrent 
fever  in  the  organism  of  immunised  guinea-pigs. — Acquired  immunity 
against  the  bacteria  of  typhoid  fever  and  pyocyanic  disease. — Acquired 
immunity  against  swine  erysipelas  and  anthrax. — Acquired  immunity 
against  the  streptococcus. — The  acquired  immunity  of  rats  against 
Trypanosoma. 

CHAPTER   IX 

THE    MECHANISM   OF   ACQUIRED   IMMUNITY   AGAINST   MICRO-ORGANISMS          .  .         250 

Cytases  and  fixatives. — Only  the  latter  are  augmented  in  the  immunised 
organism. — Properties  of  the  fixatives. — Difference  between  them  and 
the  agglutinative  substances. — The  part  played  by  the  latter  in  acquired 
immunity. — Protective  property  of  the  fluids  of  the  immunised  organism. 
—Stimulant  action  of  the  body  fluids.— The  protective  power  of  serum 
cannot  serve  as  a  measure  of  acquired  immunity. — Examples  of  acquired 
immunity  in  which  the  serums  exhibit  no  protective  power. — Phago- 


xiv  Contents 

PAGE 

cytosis  in  acquired  immunity.  —  Negative  chemiotaxis  of  leucocytes.— 
Theory  of  attenuation  of  micro-organisms  by  the  fluids  of  immunised 
animals.— Refutation  of  this  theory.— Phagocytosis  acts  without  requiring 
any  previous  neutralisation  of  the  toxins.— The  origin  of  the  fixative  and 
protective  properties  of  the  body  fluids. — The  relation  between  these 
properties  and  phagocytosis. — The  side-chain  theory  of  Ehrlich  and  the 
theory  of  phagocytes. 

CHAPTER  X 

RAPID  AND  TEMPORARY  IMMUNITY  AGAINST  MICRO-ORGANISMS,  CONFERRED  BY 
SPECIFIC  AND  NORMAL  SERUMS,  OR  BY  OTHER  SUBSTANCES,  OR  BY  MICRO- 
ORGANISMS OTHER  THAN  THOSE  AGAINST  WHICH  IT  IS  DESIRED  TO  PROTECT 
AN  ANIMAL 300 

Immunity  conferred  by  specific  serums. — Analogy  of  the  mechanism  of  this 
immunity  with  that  observed  in  immunity  obtained  with  pathogenic 
micro-organisms  and  their  products.— Part  played  by  phagocytosis  in 
the  immunity  conferred  by  specific  serums. — Influence  of  opium  on  the 
course  of  immunisation  by  these  serums. — Stimulant  action  of  specific 
serums. — Protective  and  stimulant  action  of  normal  serums. — Influence 
of  fluids,  other  than  serums :  broth,  urine,  physiological  saline  solu- 
tion, etc. 

Antagonism  between  anthrax  and  certain  bacteria. 


CHAPTER   XI 
NATURAL  IMMUNITY  AGAINST  TOXINS 325 

Examples  of  natural  immunity  against  toxins. — Immunity  of  spiders  and 
scorpions  against  tetanus  toxin. — Immunity  of  the  scorpion  against  its 
own  poison. — Anti venomous  property  of  the  blood  of  the  scorpion. — 
Immunity  against  tetanus  toxin  in  the  larvae  of  Oryctes  and  in  the 
cricket. — Immunity  and  susceptibility  of  frogs  against  this  toxin. — 
Natural  immunity  of  reptiles  against  tetanus  toxin. — Antitetanic  pro- 
perty of  the  blood  of  alligators. — Immunity  of  snakes  against  snake 
venom. — Immunity  of  the  fowl  against  tetanus  toxin. — Immunity  of  the 
hedgehog  against  poisons  and  venoms. — Immunity  of  the  rat  against 
diphtheria  toxin. 

CHAPTER  XII 
ARTIFICIAL  IMMUNITY  AGAINST  TOXINS 342 

Adaptation  to  poisons. — Artificial  immunity  against  bacterial  and  vegetable 
toxins  and  against  snake  venom. — Principal  methods  of  immunisation. — 
Immunisation  by  toxins  and  toxoids. — Inoculation  against  diphtheria 
toxin. — Phenomena  produced  in  the  course  of  vaccination  against  toxins. 
— Rise  of  temperature. — Leucocytosis. — Development  of  antitoxic  power. 
— Properties  of  antitoxins. — Mode  of  action  of  antitoxins. — Action  of 
antitoxins  in  vitro.  —  Their  action  in  the  organism.  —  Influence  of 
living  elements  on  the  combination  of  antitoxin  with  toxin. — Antitoxic 
action  of  non-specific  serums,  of  normal  serums,  and  of  broth. — Immunity 


Contents  xv 

PAGE 

against  toxins  is  not  in  direct  ratio  to  the  amount  of  antitoxins  in  the 
body  fluids.  —  Hyperseusitiveness  of  an  animal  treated  with  toxin. — 
Diminution  of  the  susceptibility  of  the  organism  immunised  against  toxins. 
Hypotheses  as  to  the  nature  and  origin  of  antitoxins.— Hypothesis  of  the 
transformation  of  toxins  into  antitoxins.  —  Hypothesis  of  receptors 
detached  from  cells  as  the  source  of  antitoxins. — Hypothesis  of  the 
nervous  origin  of  tetanus  antitoxin. — Fixation  of  tetanus  toxin  by  the 
substance  of  the  nerve  centres. — The  relations  between  saponin  and 
cholesterin. — Anti-arsenic  serum. — Part  played  by  phagocytes  in  the 
struggle  of  the  animal  against  poisons. — Probable  part  played  by  phago- 
cytes in  the  production  of  antitoxins. 

CHAPTER   XIII 

IMMUNITY  OF  THE  SKIN  AND  MUCOUS  MEMBRANES     ...        ...      403 

Protective  function  of  the  skin. — Exfoliation  of  the  epidermis  as  a  means  of 
ridding  the  animal  of  micro-organisms. — Localisation  and  arrest  of  micro- 
organisms in  the  dermis. — Intervention  of  phagocytes  in  the  defence  of 
the  skin. 

Elimination  of  micro-organisms  by  the  conjunctiva, — Microbicidal  function  of 
the  tears. — Absorption  of  toxins  by  the  conjunctiva. — Protection  of  the 
cornea. — Elimination  of  micro-organisms  by  the  nasal  mucosa. — Protection 
by  the  respiratory  channels. — Dust  cells. — Absorption  of  poisons  by  the 
respiratory  channels. 

Alleged  microbicidal  property  of  the  saliva. — Part  played  by  microbial  pro- 
ducts in  the  protection  of  the  buccal  cavity. — Antitoxic  function  of  the 
saliva. 

Antiseptic  action  of  the  gastric  juice. — Antitoxic  function  of  pepsin. 

Protective  function  of  the  alimentary  canal. — Absence  of  microbicidal  power 
from  the  intestinal  ferments. — Protective  function  of  the  bile. — Antitoxic 
rdle  of  the  digestive  ferments. — Favouring  and  retarding  functions  of 
the  intestinal  micro-organisms. — Destruction  of  toxins  by  these  micro- 
organisms. 

Defensive  role  of  the  liver.  Protective  function  of  the  lymphoid  organs  of  the 
alimentary  canal 

Protective  function  of  the  mucous  membrane  of  the  genital  organs. — Auto- 
purification  of  the  vagina. 


CHAPTER  XIV 

IMMUNITY  ACQUIRED  BY  NATURAL  MEANS 433 

Immunity  acquired  after  recovery  from  infective  diseases.  —  Immunity  ac- 
quired in  malaria. — Humoral  properties  of  convalescents  from  typhoid 
fever. — Preventive  power  of  the  blood  of  persons  who  have  recovered 
from  Asiatic  cholera. — Antitoxic  power  of  the  blood  of  persons  who 
have  recovered  from  diphtheria. 

Immunity  acquired  by  heredity. — Absence  of  hereditary  immunity  properly 
so  called. — Immunity  conferred  by  the  maternal  blood  and  by  the  yolk. 

Immunity  conferred  by  the  milk  of  the  mother. 


xvi  Contents 

CHAPTER   XV 

PAGTC 

PROTECTIVE  VACCINATIONS 454 

Vaccinations  against,  I.  Small-pox.— II.  Sheep-pox.— III.  Rabies.-IV.  Rinder- 
pest.—V.  Anthrax.— VI.  Symptomatic  Anthrax.— VII.  Swine  Erysipelas. 
-VIII.    Pleuropneumonia  in   the    Bovidae.  —  IX.    Typhoid    Fever.— 
X.  Plague.— XI.  Tetanus.— XII.  Diphtheria. 

CHAPTER  XVI 

HISTORICAL  SKETCH  OP  OUR  KNOWLEDGE  OF  IMMUNITY    .        .  .       .      505 

Methods  used  by  savage  races  for  vaccination  against  snake  venom  and  against 
bovine  pleuropneumonia. — Variolisation  and  vaccination  against  small- 
pox.— Discovery  of  the  attenuation  of  viruses  and  of  vaccinations  with 
attenuated  micro-organisms. — Theory  of  the  exhaustion  of  the  medium  as 
a  cause  of  acquired  immunity. — Theory  of  substances  which  prevent  the 
multiplication  of  the  micro-organisms  in  the  refractory  body. — Local 
theory  of  immunity. — Theory  of  the  adaptation  of  the  cells  of  the  im- 
munised organism. 

Observations  on  the  presence  of  micro-organisms  in  the  white  corpuscles. — 
History  of  phagocytosis  and  of  the  theory  of  phagocytes. — Numerous 
attacks  upon  this  theory.—  Theory  of  the  bactericidal  property  of  the 
body  fluids. — Theory  of  the  antitoxic  power  of  the  body  fluids. — Extra- 
cellular destruction  of  micro-organisms. — Analogy  between  bacteriolysis 
and  haemolysis. — Theory  of  side-chains. 

Progress  of  the  theory  of  phagocytes. — Attempts  to  reconcile  it  with  the 
humoral  theory. — Present  phase  of  the  question  of  immunity. 

CHAPTER  XVII 
SUMMARY 544 

Means  of  defence  of  the  animal  against  infective  agents. — Absorption  of  micro- 
organisms.— Phagocytes,  and  their  function  in  inflammation. — The  action 
of  phagocytes  in  the  absorption  of  micro-organisms. — The  cytases,  phago- 
cytic  ferments. — The  cytases  are  closely  bound  up  with  the  phagocytes. — 
The  fixatives  and  their  function  in  acquired  immunity. — The  fixatives  are 
excreted  by  the  phagocytes  and  pass  readily  into  the  fluids  of  the  body. 
— Essential  mechanism  of  the  action  of  the  fixatives. — Adaptation  of 
phagocytes  to  destroy  micro-organisms  in  acquired  immunity. — Difference 
between  the  fixatives  and  the  agglutinins. — Antitoxins  and  their  analogy 
with  the  fixatives. — Hypotheses  as  to  the  origin  of  antitoxins. — Cellular 
immunity  is  a  fact  of  general  import. — Susceptibility  and  its  role  in 
immunity. — Applications  of  the  theory  of  immunity  to  medical  practice. 

INDEX .  ....      571 


INTRODUCTION  [i] 

Importance  of  the  study  of  immunity  from  a  general  point  of  view. — Part  played 
by  parasites  in  infective  diseases. — Intoxications  by  the  products  of  micro- 
organisms.— Resistance  of  the  organism  to  the  invasion  of  micro-organisms. 

Natural  immunity  and  acquired  immunity. 

Immunity  to  micro-organisms  and  immunity  to  toxins. 

THE  problem  of  immunity  in  relation  to  infective  diseases  is  one 
that  not  merely  concerns  general  pathology  but  has  a  very  important 
bearing  on  all  branches  of  practical  medicine,  such  as  hygiene,  surgery 
and  the  veterinary  art.  The  prevention  of  disease  by  the  production 
of  an  acquired  immunity  is  daily  assuming  greater  importance.  With 
the  object  of  arresting  the  multiplication  and  dissemination  of 
morbific  germs,  we  are  seeking,  by  artificial  means,  to  render  indi- 
viduals, who  may  come  in  contact  with  them,  refractory  to  their 
pathogenic  action.  Patients  who  have  just  undergone  a  surgical 
operation  and  women  in  child-bed  are  frequently  in  danger  of 
acquiring  a  post-operation  disease  or  a  puerperal  affection ;  we  are, 
therefore,  striving  to  protect  them  by  conferring  upon  them  an 
artificial  immunity. 

The  immunisation  of  animals  useful  to  man  is  likewise  a  question 
of  such  great  importance  to  agriculture  and  to  industry  as  to  have 
now  become  the  object  of  legislation. 

This  question  of  immunity  is,  however,  apart  from  its  practical 
aspect,  intimately  connected  with  problems  of  pure  theory.  There 
can  be  no  question  that  the  marked  pessimism  developed  during 
the  century  just  closed  was  in  a  large  measure  prompted  by 
the  dread  of  disease  and  premature  death,  scourges  against  which 
humanity  is  as  yet  powerless.  It  is  recognised  that  Byron  and 
Leopardi,  the  great  poets  of  pessimism,  both  suffered  from  con- 
genital anomaly  and  from  incurable  disease  and  that  these  maladies 
cast  a  gloom  over  their  poetry.  Schopenhauer,  the  founder  of  the 
B.  1 


2  Introduction 

[2]  pessimistic  school  in  modern  philosophy,  was  noted  for  his  exag- 
gerated fear  of  disease. 

During  the  greater  part  of  the  nineteenth  century  our  knowledge 
as  to  immunity  has  been  limited  to  certain  practical  methods,  often 
efficacious  it  is  true,  but  purely  empirical,  such  as  those  employed  in 
immunising  man  against  small-pox  and  certain  domestic  animals 
against  sheep-pox  or  pleuro-pneumonia. 

So  long  as  the  nature  of  the  viruses  was  unknown  no  really 
scientific  study  of  their  action  or  of  immunity  from  them  could  be 
made.  The  revelation  of  the  organised  nature  of  the  infective  viruses 
opened  up  the  way  for  these  researches.  This  discovery,  the  out- 
come of  the  demonstration  by  Pasteur  of  the  organised  nature  of 
the  ferments,  has  enabled  us  to  establish  the  part  played  by  living 
agents  in  a  great  number  of  infective  diseases,  and,  linked  with  the 
names  of  Davaine,  Obermeyer,  and  above  all  with  that  of  Robert 
Koch,  it  has  very  greatly  advanced  the  study  of  susceptibility  and  of 
natural  immunity  in  certain  infections. 

A  considerable  forward  step  was  made  with  the  discovery,  by 
Pasteur  and  his  collaborators  Chamberland  and  Roux,  that  it  was 
possible,  in  certain  infective  diseases,  to  confer  immunity  by  means  of 
micro-organisms  which  had  had  their  virulence  attenuated.  Thanks 
to  this  discovery,  science  was  now  in  a  position  to  take  up  the 
thorough  study  of  acquired  immunity.  The  field  of  research  was  still 
further  enlarged  by  the  demonstration  of  the  immunising  power  of 
the  culture-products  of  pathogenic  micro-organisms  and  above  all 
by  the  discovery  that  the  blood  of  immunised  animals  is  capable  of 
conferring  immunity  upon  susceptible  animals. 

Before  taking  up  in  detail  the  problem  of  immunity  as  it  is 
revealed  to  us  as  a  consequence  of  these  discoveries,  it  is  essential  to 
cast  a  glance  at  infective  and  allied  diseases  as  a  whole  and  to  indicate 
in  what  light  we  look  upon  them  in  view  of  the  present  state  of  our 
knowledge. 

It  has  been  definitely  established  that  many  infective  diseases 
of  man  and  animals  are  due  to  the  invasion  of  small  parasitic 
organisms,  sometimes  of  animal  nature  (as  in  itch,  trichinosis,  malaria, 
Texas  fever,  nagana,  or  surra  and  the  allied  condition  "  dourine  "  in 
horses),  sometimes  belonging  to  the  vegetable  kingdom  like  the 
Moulds  (aspergillosis),  the  Hyphomycetes  (actinomycosis,  Madura  foot 
[3]  disease,  bovine  farcy)  and  the  Yeasts  (disease  of  the  Daphniae,  some 
pseudomyxomas  and  septicaemias,  pseudolupus).  But  by  far  the 


Introduction  3 

greater  number  of  infective  diseases  are  due  to  the  development  in 
the  organism  of  plants  of  the  simplest  structure,  Bacteria.  These 
Bacteria  produce  the  gravest  and  most  destructive  infections,  such 
as  tuberculosis,  bubonic  plague,  diphtheria,  cholera,  anthrax,  the 
pneumonias,  suppuration,  erysipelas,  tetanus,  glanders,  leprosy,  &c. 
Among  these  bacteria  some  are  too  small  to  be  resolved  individually 
under  the  highest  magnifying  powers  and  can  only  be  made  out  en 
masse.  Such  is  the  micro-organism  of  the  contagious  pleuro-pneumonia 
of  cattle.  To  this  minuteness  of  certain  pathogenic  Bacteria  is  very 
probably  due  the  fact  that  in  a  considerable  number  of  infections, 
amongst  which  are  scarlatina,  measles,  rabies,  syphilis,  aphthous  fever 
and  small-pox,  it  has  been  impossible,  up  to  the  present,  to  recognise 
any  specific  micro-organisms. 

It  is  probable  that  we  shall  succeed  in  discovering  parasites,  not 
only  in  the  diseases  I  have  just  cited,  which  present  the  characters  of 
infective  and  virulent  diseases,  but  also  in  diseases  of  entirely  different 
types.  In  spite  of  the  failure  of  various  attempts  to  demonstrate  the 
parasite  of  malignant  tumours,  it  may  be  hoped  that,  with  improvement 
in  scientific  methods,  such  a  parasite  will  be  unequivocally  demon- 
strated. In  many  other  conditions  which  are  at  present  considered 
as  not  dependent  on  micro-organisms,  an  intimate  connection  with 
such  organisms  will  probably  be  established.  Such  are  the  atrophic 
diseases  and  certain  diseases  of  nutrition  in  which  the  parasites,  with- 
out playing  a  direct  or  immediate  role,  act  by  means  of  their  products, 
or  by  the  changes  which  they  set  up  in  the  affected  organism.  To 
give  an  idea  of  this  possibility  it  will  be  useful  to  cast  a  glance  at  the 
various  modes  of  action  of  the  numerous  etiological  agents  in  infective 
diseases.  The  parasites  which  produce  them  have,  as  a  common 
feature,  their  small  dimensions ;  they  can  only  be  recognised  with 
precision  by  the  employment  of  high  powers  of  the  microscope. 
They  are  likewise  distinguished  by  a  great  variability,  which  is  not 
astonishing,  since  among  infective  agents  are  found  on  the  one  hand 
animals  of  high  structure  (such  as  the  Acari  of  itch)  and  on  the  other 
plants  of  the  simplest  character  such  as  the  Gonococci  or  the  various 
Cocco-bacilli. 

The  Acari  are  capable  of  perforating  the  epidermis  by  the  mech-  [4] 
anical  action  of  their  feet  and  mouth-parts.    They  excavate  channels 
in  the  skin  and  thus  provoke  the  irritation  so  characteristic  of  itch. 
The  larvae  of  the  Trichinae  in  like  manner  produce  marked  lesions  by 
the  mere  mechanical  act  of  penetration  and  migration  in  the  striped 

1—2 


4  Introduction 

fibre  of  muscular  tissue.  In  human  trichinosis,  however,  the  disease 
picture  is  more  complicated  than  in  itch  and  leads  us  to  assume  that 
there  is  some  additional  action  of  the  excreta  of  the  larvae  in  the 
production  both  of  the  febrile  state  and  of  certain  general  phenomena. 
In  the  nagana  disease  (transmitted  by  the  Tsetse  fly)  there  is  equal 
reason  to  admit  the  preponderating  role  of  the  ^mechanical  action]  of 
the  flagellated  parasite  (Trypanosoma)  which  obstructs  the  vessels 
of  the  nervous  centres. 

In  the  diseases  which  are  set  up  by  Fungi,  such  as  ringworm  and 
aspergillosis,  the  purely  mechanical  element  still  appears  to  play  the 
more  important  part.  Even  certain  of  the  bacterial  infections  mani- 
fest this  same  character.  Thus,  there  is  no  doubt  that  in  chronic 
tuberculosis  in  the  guinea-pig,  Koch's  bacillus  brings  about  a  substi- 
tution of  tuberculous  elements  for  the  normal  tissues,  and  this  to 
such  a  degree  that,  at  the  termination  of  the  disease,  there  may 
remain  merely  traces  of  the  liver  and  of  the  lungs,  and  the  animal 
dies  for  want  of  these  organs,  whose  normal  action  is  no  longer 
possible.  In  the  tuberculous  guinea-pig  the  phenomenon  of  intoxica- 
tion by  the  bacillary  poisons  plays  but  a  secondary  role;  yet  there 
are  examples  of  tuberculosis  (as  in  acute  miliary  tuberculosis  in  man 
or  experimental  tuberculosis  in  cattle,  obtained  by  Nocard's  method 
of  inoculation  into  the  milk  ducts),  where  the  poisoning  assumes 
much  greater  importance. 

Among  the  bacterial  diseases  of  man,  leprosy  may  be  cited  as  one 
in  which  the  intoxication  is  relegated  to  a  subsidiary  position,  yielding 
place  to  the  mechanical  substitution  of  the  specific  granuloma  for  the 
normal  tissues.  It  is  only  in  the  acute  leprous  exacerbations  that  we 
perceive  any  signs  of  intoxication  by  the  products  of  the  leprosy 
bacillus. 

All  the  instances  cited,  however,  constitute  but  a  feeble  minority 
which  is  completely  thrown  into  the  shade  in  the  presence  of  very 
numerous  infections  in  which  the  toxic  element  dominates  the  situation. 
Even  in  carbuncular  diseases  an  exact  analysis  of  their  morbid  phe- 
nomena has  compelled  us  to  recognise  the  marked  influence  of  the 
[5]  poison  produced  by  the  bacterium.  The  majority  of  the  micro- 
organisms act  as  poisoners  which  introduce  themselves  into  the 
organism  where  they  can  secrete  toxins  capable  of  provoking  general 
disorders  of  very  diverse  natures.  Indeed  in  infective  diseases  a  whole 
gamut  of  very  remarkable  variations  is  produced.  Thus  many  of  the 
micro-organisms  capable  of  setting  up  septicaemias  must  multiply 


Introduction  5 

abundantly  in  the  organism  and  be  distributed  in  the  blood,  before 
they  can  produce  a  general  morbid  condition.  The  spirillum  of 
human  recurrent  fever  is  an  example  of  this.  It  multiplies  for  some 
days  and  produces  several  generations  without  provoking  the  least 
malaise  ;  then,  however,  their  appearance  in  the  blood  suddenly 
produces  intense  fever  and  constitutional  phenomena  of  the  most 
pronounced  character. 

On  the  other  hand  there  are  certain  bacteria  which  are  dis- 
tinguished by  a  very  much  feebler  reproductive  power,  but  a  more 
marked  toxic  activity.  Incapable  of  spreading  through  the  organism, 
these  bacteria  remain  localised  at  the  point  of  entrance,  where  they 
secrete  their  poisons  and  thus  frequently  set  up  a  fatal  intoxication. 
Some  of  these  bacteria,  such  as  the  bacilli  of  tetanus  and  of  diphtheria, 
penetrate  more  or  less  deeply  into  the  living  tissues  of  the  affected 
animal.  Others  can  manifest  their  toxic  action  so  to  speak  at  a 
distance  or  by  simple  contact  with  the  living  elements.  Into  this 
category  comes  the  organism  of  Asiatic  cholera.  Koch's  vibrio,  once 
established  in  the  intestine,  there  secretes  its  poison ;  this,  absorbed 
by  the  apparently  intact  intestinal  mucous  membrane,  sets  up  a  fell 
disease,  purely  toxic  in  character.  It  is  probable  that  in  the  case  of 
those  intestinal  diseases  whose  etiology  is  still  unknown,  such  as 
infantile  choleras,  the  poisoning  by  the  products  of  micro-organisms 
constitutes  the  essential  phenomenon.  The  micro-organisms  do  not 
make  their  way  into  the  blood  or  tissues ;  they  remain  in  the  contents 
of  the  intestine  and  thence  set  up  their  deadly  intoxication. 

Instances  do  exist  in  which  the  pathogenic  micro-organism  disap- 
pears from  the  body,  leaving  there  a  toxin  which,  alone,  is  responsible 
for  death.  Thus  in  the  spirillar  septicaemia  of  geese,  the  birds  die 
at  a  stage  when  not  a  single  living  spirillum  can  be  found  in  the 
body.  The  poisoners  have  been  destroyed  before  the  toxin  produced 
by  them  had  completed  its  work.  In  other  instances,  e.g.  typhoid  fever 
of  the  horse,  the  specific  micro-organism  likewise  disappears  before 
the  death  of  the  animal ;  but  at  the  period  when  the  poison  of  this 
bacterium  finishes  its  fatal  work,  there  is  a  secondary  invasion  of  [6] 
other  micro-organisms  which  have  nothing  to  do  with  the  typhoid 
fever  proper  of  the  horse. 

This  great  variability  in  the  action  of  the  different  pathogenic 
agents  is  still  further  increased  through  the  differing  relations  between 
the  parasites  and  the  affected  organism.  Certain  micro-organisms 
are  capable  of  producing  a  typical  disease,  whatever  may  be  the 


6  Introduction 

mode  and  seat  of  invasion  of  the  organism.  But  these  are  compara- 
tively few  in  number.  The  bacillus  of  tuberculosis  belongs  to  this 
minority.  Whether  it  enters  subcutaneously,  by  the  eye  or  by  the 
respiratory,  digestive  or  genito-urinary  passages,  it  invariably  pro- 
duces tubercular  lesions  more  or  less  grave  and  more  or  less  capable 
of  generalisation.  On  the  other  hand,  a  very  large  number  of  micro- 
organisms only  exert  their  pathogenic  action  when  they  invade  the 
organism  at  definite  points.  The  anthrax  bacillus,  when  introduced 
through  the  slightest  lesion  of  the  skin  or  of  the  mucous  membranes, 
produces  in  man,  and  in  a  large  number  of  mammals,  a  very  grave 
and  usually  fatal  disease ;  when  absorbed  in  the  vegetative  state  with 
food,  it  is  almost  always  innocuous.  With  the  cholera  vibrio  we 
have  an  exactly  opposite  condition  of  affairs.  When  inoculated,  even 
in  large  numbers,  below  the  skin  in  the  human  subject,  it  rapidly 
disappears,  producing  merely  insignificant  disturbances ;  but  when 
the  same  vibrio  is  introduced  into  the  digestive  canal  it  develops  and 
produces  Asiatic  cholera,  a  disease  so  often  terminating  in  death. 

All  these  variations  and  peculiarities  associated  with  the  nature  of 
infective  agents  are  of  great  importance  from  the  point  of  view  of 
immunity. 

Do  diseases  come  from  without  or  do  their  causes  arise  within  the 
organism?  is  a  pressing  question,  long  discussed  by  pathologists. 
Those  who  have  discovered  most  of  the  pathogenic  micro-organisms 
have  ranged  themselves  on  the  side  of  the  former  hypothesis.  For 
the  majority  of  them  the  essential  etiological  factor  in  the  causation 
of  infective  diseases  consists  in  the  invasion  of  the  patient  by  the 
pathogenic  micro-organism  from  the  outer  world.  This  theory  is  in 
perfect  harmony  with  many  of  the  admitted  facts  of  epidemiology, 
according  to  which  the  viruses  of  the  most  deadly  epidemic  diseases, 
such  as  Asiatic  cholera,  yellow  fever,  and  bubonic  plague,  must  be 
imported  into  a  country  previously  free  from  the  disease  before  an 
epidemic  can  be  developed.  In  anthrax  and  trichinosis  it  is  recognised 
[7]  that  the  parasites  must  come  from  without.  Hence,  in  the  study  of 
,  pathogenic  micro-organisms  one  always  follows  the  rule  that  it  is 
essential  ta  find  the  specific  micro-organism  in  all  cases  of  the  disease 
in  'question  and  to  prove  its  absence  in  healthy  individuals  or  in 
those  who  are  affected  with  other  diseases.  Thus,  Koch  *,  in  his  classical 
researches  on  Asiatic  cholera,  insisted  on  the  fact  -that  the  cholera 
vibrio  was  always  found  in  cases  of  this  disease  but  never  in  healthy 
1  Deutsche  med.  Wchnschr.,  Leipzig,  1884,  SS.  499,  519. 


Introduction  7 

persons.  Almost  simultaneously  Loeffler1,  in  the  course  of  his  work 
on  the  etiology  of  diphtheria,  demonstrated  the  presence  of  a  specific 
bacillus  not  only  in  a  large  number  of  cases  of  this  disease  but  also  in 
the  throat  of  a  healthy  child;  and  this  fact  at  first  prevented  him 
from  accepting  this  bacillus  as  the  real  cause  of  diphtheria. 

This  view  accepted  by  two  such  eminent  bacteriologists  cannot 
however  be  maintained.  It  is  impossible  to  assume  that  each  time 
that  a  pathogenic  micro-organism  makes  its  way  into  a  susceptible 
species  its  presence  must  inevitably  be  followed  by  the  production  of 
the  specific  disease.  Although  the  discovery  by  Loeffler  of  the 
diphtheria  bacillus  in  the  throat  of  healthy  individuals  has  repeat- 
edly been  confirmed,  it  is  impossible  to  doubt  the  etiological  role  of 
this  organism  in  diphtheria.  Moreover,  it  has  been  established  that 
Koch's  vibrio,  although  undoubtedly  the  etiological  factor  in  the 
production  of  Asiatic  cholera,  has  nevertheless  been  recognised  in  the 
digestive  canal  of  perfectly  healthy  persons. 

As  soon  as  he  is  born,  man  becomes  the  habitat  of  a  very  rich 
microbial  flora.  The  skin,  the  mucous  membranes,  and  the  gastro- 
intestinal contents  become  stocked  with  such  a  flora,  but  a  very  small 
number  of  these  micro-organisms  have  up  to  the  present  been  recog- 
nised or  described.  The  buccal  cavity,  the  stomach,  the  intestines 
and  the  genital  organs  offer  a  feeding  ground  for  Bacteria  and 
inferior  Fungi  of  various  kinds.  For  long  it  was  thought  that  in 
healthy  individuals  all  these  micro-organisms  were  inoffensive  and 
sometimes  even  useful.  It  was  supposed  that  when  an  infective 
malady  was  set  up  a  specific  pathogenic  micro-organism  was  added  to 
the  benign  flora.  Exact  bacteriological  researches  have,  however, 
clearly  demonstrated  that  as  a  matter  of  fact  the  varied  vegetation  in 
healthy  persons  often  includes  representatives  of  noxious  species  of  [8] 
bacteria.  Besides  the  diphtheria  bacillus  and  the  cholera  vibrio,  which 
have  repeatedly  been  found  in  a  virulent  form  in  perfectly  healthy 
individuals,  it  has  been  demonstrated  that  certain  pathogenic  micro- 
organisms, e.g.  the  Pneumococcus,  staphylococci,  streptococci  and  the 
Bacillus  coli,  are  always,  or  almost  constantly,  found  among  the 
microbial  flora  of  healthy  persons. 

This  observation  has  necessarily  led  to  the  conclusion  that  in 
addition  to  the  micro-organism   there  exists  a  secondary  cause  of 
infective  diseases — a  predisposition,  or  absence  of  immunity.    An  in- 
dividual in  whom  one  of  the  above-mentioned  pathogenic  species  is 
1  Mitth.  ausd.  K.  Gesundheitsamte,  Berlin,  1884,  Bd.  11.  S.  421. 


8  Introduction 

present,  manifests  a  permanent  or  transitory  refractory  state  as  regards 
this  specific  organism.  As  soon  however  as  the  cause  of  this  immunity 
ceases  to  act,  the  micro-organism  gets  the  upper  hand  and  sets  up  the 
specific  disease.  It  is  thus  in  diabetic  persons  that  boils  make  their  ap- 
pearance as  the  result  of  the  development  of  Staphylococcus  pyogenes, 
a  micro-organism  that  is  almost  always  found  in  abundance  on  the 
skin  and  mucous  membranes  of  the  human  subject.  The  diabetes  is, 
in  these  cases,  the  cause  of  the  suspension  of  the  immunity  which 
exists  in  the  healthy  individual. 

People  who  carry  the  Pneumococcus  on  their  mucous  membranes 
may  remain  for  long  without  being  attacked  by  fibrinous  pneumonia 
or  any  of  the  other  maladies  due  to  this  micro-organism.  But  often, 
in  consequence  of  some  special  circumstance,  a  cold  for  example,  the 
refractory  state  gives  way  to  a  more  or  less  marked  susceptibility. 

It  is  unnecessary  to  multiply  the  number  of  such  examples;  they 
demonstrate  in  the  clearest  fashion  that,  in  addition  to  the  causes 
of  disease  which  come  from  the  outer  world  and  which  are  represented 
by  the  micro-organisms,  there  are  yet  other  causes  which  lie  within 
the  organism  itself.  When  these  internal  factors  are  powerless  to 
prevent  the  development  of  the  morbific  germs,  a  disease  is  set  up ; 
when,  on  the  other  hand,  they  resist  the  invasion  of  the  micro- 
organisms properly,  the  organism  is  in  a  refractory  condition  and 
exhibits  immunity. 

Diseases  in  general  and  infective  diseases  in  particular  were 
developed  on  the  earth  at  a  very  remote  epoch.  Far  from  being 
peculiar  to  man,  animals  and  the  higher  plants,  they  attack  inferior 
forms  and  are  widely  distributed  among  unicellular  organisms,  In- 
[9]  fusoria  and  Algae.  Diseases  undoubtedly  play  an  important  role  in 
the  history  of  life  on  our  planet,  and  it  is  very  probable  that  they 
have  contributed  in  a  marked  degree  to  the  extinction  of  certain 
species.  When  we  observe  the  ravages  produced  by  parasitic  Fungi 
among  the  young  fish  which  we  are  trying  to  rear,  or  the  destruction 
of  crayfish  in  certain  countries  in  consequence  of  the  rapid  increase 
of  epizootic  germs,  we  are  involuntarily  led  to  the  conclusion  that 
pathogenic  micro-organisms  must  have  brought  about  the  disappear- 
ance of  certain  animal  and  vegetable  species. 

Darwin1,  in  the  chapter  on  the  extinction  of  species  in  his  book 
On  the  Origin  of  Species,  states  upon  the  authority  of  several 
observers  that  insects  so  annoy  elephants  that  these  large  mammals 
1  "  On  the  Origin  of  Species,"  6th  ed.,  London,  1872,  Chapter  xr,  p.  277. 


Introduction  9 

become  incapable  of  reproducing  themselves  in  sufficient  numbers. 
Xow  it  is  proved  that  many  Insects  inoculate  pathogenic  micro- 
organisms and  thus  transmit  destructive  diseases.  A  most  for- 
midable epizootic  disease,  provoked  by  a  flagellated  Infusorium,  the 
Trypanosoma  brucei,  is  inoculated  into  large  mammals  in  South 
Africa  by  a  fly,  the  Tsetse  fly;  in  certain  districts  this  disease  is  so 
widespread  and  so  destructive  that  the  rearing  of  domestic  animals 
becomes  impossible. 

Parasites  strike  then  with  great  intensity,  bringing  about  the 
destruction  of  numerous  human  beings,  animals  and  plants.  Never- 
theless, in  spite  of  the  disappearance  of  a  large  number  of  species, 
the  world  continues  well  populated.  This  fact  proves  that,  by  the 
special  means  at  the  disposal  of  the  organism,  without  any  aid  of  the 
medical  art  or  special  human  intervention,  many  living  species  have 
held  their  own  throughout  the  ages.  Everybody  has  seen  how  dogs 
lick  their  wounds,  moistening  them  with  a  saliva  full  of  micro- 
organisms. These  wounds  heal  well  and  quickly  without  dressings 
or  antiseptics. 

In  all  these  examples  the  resistance  of  the  organism  depends 
on  immunity,  a  condition  very  general  in  nature.  This  immunity 
against  infective  diseases  is  very  complex  and  its  thorough  study 
could  only  be  undertaken  after  we  had  acquired  an  extended  knowledge 
of  these  diseases,  and  after  adequate  methods  of  research  had  been 
devised. 

By  immunity  against  infective  diseases  we  understand  the  re-  [10] 
sistance  of  the  organism  against  the  micro-organisms  which  cause 
these  diseases.  We  have  here  to  do  with  an  organic  property  of 
living  beings  and  not  with  the  immunity  which  belongs  to  certain 
countries  or  localities.  For  this  reason  information  on  the  causes 
of  the  immunity  in  Europe  and  in  mountainous  regions  from  yellow 
fever  will  not  be  found  in  this  book,  nor  why  the  majority  of 
Europeans  do  not  take  recurrent  fever.  The  inhabitants  of  our 
continent  do  not  possess  organic  immunity  against  either  the  virus 
of  yellow  fever  or  Obermeyer's  spirillum  of  recurrent  fever.  Indeed 
they  are  very  susceptible  to  these  diseases.  It  is  solely  the  con- 
ditions of  life,  in  the  majority  of  European  countries,  that  prevent 
the  invasion  by  the  specific  germs  and  the  creation  of  epidemic 
foci.  The  same  point  of  view  ought  also  to  be  applied  to  animals. 
Our  small  laboratory  rodents,  mice  and  guinea-pigs,  are  much  more 
susceptible  to  anthrax,  whether  inoculated  beneath  the  skin  or 


10  Introduction 

in  any  other  part  of  the  body,  than  are  the  large  domestic  mammals 
such  as  the  ox  and  the  horse.  And  yet  these  latter  are  very 
liable  to  epizootic  anthrax,  whilst  the  rodents  mentioned  are  seldom, 
if  ever,  attacked  by  spontaneous  anthrax.  This  apparent  immunity 
in  no  way  depends  on  the  existence  of  a  true  immunity  of  the 
organism,  but  solely  on  the  conditions  under  which  mice  and  guinea- 
pigs  live. 

We  shall  therefore  in  this  volume  treat  only  of  the  phenomena  of 
organic  immunity  in  living  beings,  and  the  problem,  even  restricted 
within  these  limits,  still  appears  sufficiently  complex.  With  the 
object  of  rendering  its  study  as  easy  as  possible,  it  will  be  useful  to 
commence  by  giving  an  account  of  the  phenomena  of  immunity  in  the 
lowest  organisms. 

Immunity  against  infective  diseases  should  be  understood  as  the 
group  of  phenomena  in  virtue  of  which  an  organism  is  able  to  resist 
the  attack  of  the  micro-organisms  that  produce  these  diseases.  It 
is  impossible,  at  present,  to  give  a  more  precise  definition,  and 
useless  to  insist  upon  it.  Some  have  thought  it  necessary  to  dis- 
tinguish between  immunity  properly  so  called,  that  is  to  say  a 
permanent  refractory  state,  and  "resistance,"  or  a  very  transient 
property  of  opposing  the  invasion  of  certain  infective  micro-organisms. 
It  is  not  possible  to  maintain  this  distinction,  for  in  reality  the  limits 
between  these  two  groups  of  phenomena  are  far  from  being  constant. 
[11]  Immunity  may  be  inborn  or  acquired.  The  former  is  always 
natural,  that  is  to  say,  independent  of  the  direct  intervention  of 
human  art.  Acquired  immunity  is  also  often  natural,  from  the  fact 
that  it  is  established  as  the  result  of  the  spontaneous  cure  of  an 
infective  disease.  But  in  a  great  number  of  cases  acquired  immunity 
may  be  the  result  of  direct  human  intervention  as  in  the  practice 
of  vaccination. 

For  a  long  time  all  the  phenomena  of  immunity  against  infective 
diseases  were  collected  into  a  single  group.  Later,  it  was  recognised, 
as  the  result  of  the  demonstrations  summarised  at  the  beginning  of 
this  chapter,  that  it  is  necessary  to  distinguish  sharply  between 
immunity  against  the  pathogenic  micro-organisms  themselves  and 
that  against  microbial  poisons.  Hence  the  idea  of  antimicrobial 
and  antitoxic  immunities.  In  the  course  of  this  work  this  essential 
distinction  must  always  be  borne  carefully  in  mind. 


CHAPTER  I 
IMMUNITY  IX  UXICELLULAR  ORGANISMS 

Infective  diseases  of  unicellular  organisms. — Intracellular  digestion  in  the  Protozoa. —  [13] 
Amoebo-diastase. — Part  played  by  digestion  in  the  defence  of  the  Protozoa 
against  infective  parasites. — Defences  oftheParamaecia  against  micro-organisms. 
— Part  played  by  irritability  in  defence  in  the  lower  organisms. 

Immunity  of  unicellular  organisms  to  toxins. — Acclimatisation  of  Bacteria  to  toxic 
substances. — Protective  secretion  of  membranes  by  Bacteria. 

Adaptation  of  the  Protozoa  to  saline  solutions — of  yeasts  to  poisons — of  yeasts 
to  milk-sugar. 

Irritability  of  unicellular  organisms  and  Weber- Fechner's  psycho-physical  law. 

THE  immunity  of  unicellular  organisms  against  infective  diseases  and 
against  toxic  agents  is  as  yet  very  imperfectly  understood.  Never- 
theless, it  will  be  very  useful  for  us  to  begin  our  study  of  the  problem 
of  immunity  on  these  lower  organisms,  because  of  their  greater 
general  simplicity.  It  may  be  affirmed  that  if  the  line  of  comparative 
pathology  had  been  followed  in  our  study  of  the  etiology  of  diseases 
of  man  and  the  higher  animals,  the  parasitic  nature  of  these  infections 
would  have  been  established  considerably  earlier  than  was  the  case. 
Thus,  at  a  period  when  medical  men  and  veterinary  surgeons  were 
content  to  record  the  presence  of  Bacteria  in  the  blood  of  their 
patients,  without  attributing  to  them  the  slightest  etiological  role, 
botanists  and  zoologists  had  already  proved  most  definitely  that 
many  plants  and  lower  animals  were  subject  to  epidemic  diseases 
undoubtedly  set  up  by  the  parasitism  of  various  exceedingly  simple 
organisms.  In  the  same  year,  1855,  that  Pollender1  published  his  first 
observations  on  the  bacterium  found  in  the  blood  of  animals  affected 
by  anthrax  though  he  could  not  trace  the  slightest  relation  between 
the  presence  of  this  organism  and  the  etiology  of  the  disease,  the 

1  Vrtljschr.f.gerichtl.  Med.}  Berlin,  1855,  S.  102. 


12  Chapter  I 

celebrated  botanist  Alexander  Braun1  issued  his  work  on  the  genus 
[14]  Chytridium,  in  which  he  demonstrated  the  fact  that  certain  plants 
and  flagellated  Infusoria  suffer  from  the  invasion  of  a  small  mobile 
parasite  which,  attaching  itself  to  their  body-wall,  absorbs  the 
contents  and  so  destroys  its  hosts,  causing  a  very  great  mortality 
among  them.  The  cycle  of  development  in  the  Chytridia,  established 
by  Braun,  left  no  doubt  as  to  the  accuracy  of  his  view  and  even 
renders  it  possible  for  us  to  interpret  more  accurately  the  earlier 
observations  of  Stein,  on  the  supposed  evolution  of  certain  Infusoria, 
by  showing  that  the  changes  observed  in  these  organisms  were  in 
reality  due  to  an  invasion  by  Chytridia. 

Since  these  observations  were  made  it  has  been  clearly  demon- 
strated that  among  the  unicellular  organisms,  certain  Flagellata  and 
ciliated  Infusoria  are  subject  to  infective  maladies  the  result  of 
parasitism  of  the  Chytridiaceae,  a  group  of  the  lower  Fungi.  Small, 
mobile,  colourless  cells  attach  themselves  to  the  surface  of  the 
Protozoa,  penetrate  into  their  interior  and  absorb  the  greater  part 
of  their  living  content.  Sometimes  these  parasites  multiply  in  a  most 
extraordinary  fashion  and  destroy  enormous  numbers  of  the  Infusoria. 
Thus,  Nowakowski2,  who  has  given  a  very  detailed  description  of 
Polyphagus  euglenae,  the  Chytridium  of  the  common  green  fresh- 
water Euglena,  records  the  disappearance  of  the  Euglenae  from 
his  aquaria  glasses :  the  parasites  "  were  reproduced  in  such  great 
abundance  that  ultimately  they  had  completely  replaced  the  Euglenae." 
The  Flagellata,  subject  to  infection  by  Chytridict,  are  found  almost 
exclusively  amongst  those  genera  (Cryptomonas,  Chlamydomonas, 
Haematococcus,  Phacus,  Volvox,  etc.)  which  are  nourished  after  the 
fashion  of  vegetables,  that  is  by  the  absorption  of  substances  dissolved 
in  the  surrounding  fluids.  It  is  very  remarkable  that  in  the  group  of 
ciliated  Infusoria  this  parasitism  of  the  Chytridia  is  observed  almost 
solely  in  the  encysted  forms,  that  is  to  say,  at  a  stage  when  the 
animalcules,  surrounded  by  their  envelope,  do  not  take  any  nourish- 
ment. The  invasion  by  the  Chytridia  has  been  demonstrated  in  the 
case  of  the  cysts  of  the  Vorticellina,  Oxytrichinina,  Nassula,  etc.3 
These  facts  indicate  that  the  absence  of  the  digestion  of  solid 
aliments,  such  as  occurs  in  almost  all  the  ciliated  Infusoria,  con- 

1  "Ueber  Chytridium,"  in  Mcmatsber.  d.  Berliner  Akad.,  1855,  June,  No.  14. 

2  Conn's  "Beitrage  zur  Biologic  der  Pflanzen,"  Breslau,  1876,  Bd.  n,  S.  210. 

3  For  the  parasites  of  Infusoria,  cf.  Biitschli  in  Bronn's  "  Klassen  und  Ordnungen 
d.  Thier-Reichs,"  Leipzig,  1885—1889,  Bd.  i,  SS.  872,  1823,  1944. 


Immunity  in  Unicellular  Organisms  13 

stitutes  a  condition  favourable  to  infection  by  the  Chytridia.    Whilst  [15] 
the  growth  of  Volvocina,  Euglenae  and  their  allies  is  almost  always 
interfered  with  by  very  destructive  parasitic  epidemics,  the  ciliated 
Infusoria,  capable  of  seizing  and  digesting  lower  organisms,  may  be 
cultivated  and  flourish  for  a  very  long  period.    Thus  Balbiani1  has 
watched  one  of  his  cultures  of  Paramaecium  aurelia  multiply  and 
thrive  in  splendid  condition  for  14  years  in  succession.    Now  these 
Infusoria  readily  adapt  themselves  to  ordinary  water  untreated  to 
render  it  more  hygienic.    Such  water  swarms  with  all  sorts  of  lower 
organisms,  among  which  are  the  Chytridia  and  numerous  Bacteria,  but 
the  Paramaecia  and  Infusoria  in  general  feed  upon  these  organisms 
and  contribute  largely  to  the  purification  of  the  water.    Almost  the 
whole  body-contents  in  a  ciliated  Infusorian  is  made  up  of  a  digestive 
protoplasm    into  which    the    captured   Bacteria   and   other   lower 
organisms    are    conveyed ;    the    nutrient    particles    becoming    sur- 
rounded by  transparent  vacuoles,  in  which  the  ingested  organisms 
are  killed  and  digested.  The  food  contained  in  the  vacuoles  circulates 
in   the   endoplasm    of   the    Infusoria  by  means  of  the   streaming 
movements  of  this  layer.    The  digestive  vacuoles  become  filled  with 
a  fluid  having  a  distinctly  acid  reaction.    Formerly,  in  order  to 
demonstrate   this  reaction,  Infusoria  were  allowed  to  ingest   small 
granules   of  blue  litmus  which   after  a  certain  time  became  more 
or  less  intensely  red ;   but  the   use  of  aniline  colours  has  much 
simplified    the    study  of   digestion   in  microscopic  organisms.    By 
introducing  a  solution  of  alizarin  sulpho-acid  into  a  liquid  con- 
taining Infusoria,   the   yellow  staining   (characteristic   of  the  acid 
reaction)  of  the  digestive  vacuoles  can  be  readily  made  out.    When 
the  Infusoria  ingest  small  clumps  of  alkaline  substances,  stained 
violet  by  this  reagent,  the  vacuoles  take  on  a  red  tint,  indicating 
the  acidity  of  their  contents2.    Another  aniline  colour,  neutral  red 
(Xeutralroth),  introduced  into  microscopical  technique  by  Ehrlich3, 
enables  us  to  demonstrate  the  acid  reaction  in  the  digestive  vacuoles 
even  within  a  few  minutes.  Thus,  in  Paramaecia  treated  with  a  dilute 
solution  of  this  reagent,  the  digestive  vacuoles  at  once  assume  the 
deep  rose  tint,  characteristic  of  an  acid  reaction.    This  coloration  is 
observed  during  the  life  of  the  Infusorian,  but  immediately  after  death  [16] 

1  Arch,  d'anat.  microsc.,  Paris,  1898,  t.  u,  p.  528. 

2  Le  Dantec,  "Recherches  sur  la  digestion  intracellulaire,"  Lille,  1891,  p.  53. 

3  Ehrlich  u.  Lazarus,  "Die  Anamie,"  in  Nothnagel's  "Specielle  Pathologic  u. 
Therapie,"  Wien,  1898,  Bd.  vm,  Iter  Theil,  S.  85;  also  "Pathology  of  the  Blood," 
authorised  English  translation,  Cambridge,  1900,  p.  125. 


14  Chapter  I 

the  vacuoles  become  brownish  and  then  completely  lose  their  colour. 
This  reaction,  easily  demonstrated,  indicates  that  neutralisation  of  the 
acid  of  the  vacuoles  by  the  protoplasm  and  the  surrounding  water, 
both  of  which  are  alkaline  in  reaction,  has  taken  place. 

In  a  medium  distinctly  acid  the  Infusoria  digest  their  prey  which, 
in  a  very  great  number  of  cases,  consists  of  Bacteria.  These  micro- 
organisms are  swallowed  and  carried  into  the  digestive  endoplasm 
in  the  living  condition ;  we  have  evidence  of  this  in  the  active 
movements  of  a  certain  number  of  the  bacteria ;  at  first  they  are 
found  isolated  in  the  interior  of  the  vacuoles,  but  later  they  collect 
into  more  or  less  compact  clumps.  These  masses  of  micro-organisms 
undergoing  digestion,  when  treated  with  neutral  red  assume  a  very 
deep  rose  tint,  preserving  their  bacillary  form  to  the  end,  that  is  to 
say  up  to  the  extrusion  of  the  effete  or  waste  material.  There  is, 
indeed,  only  very  imperfect  dissolution  not  only  of  the  bacilli  as  a 
whole  but  also  of  their  contents.  Paramaecia  placed  amongst 
cholera  vibrios  swallow  them  greedily  and  in  great  numbers, 
digesting  them  as  they  would  any  other  micro-organism.  I  have 
never  been  able  to  see  any  conversion  of  vibrios  into  granules  going 
on  within  the  digestive  vacuoles. 

All  the  attempts  that  have  been  made  in  my  laboratory  to  extract 
a  digestive  fluid  from  Paramaecia  have  failed  entirely.  Very 
large  quantities  of  these  Infusoria,  obtained  by  filtration  of  rich 
cultures,  and  macerated  by  different  methods,  have  proved  inactive 
even  in  the  case  of  those  Bacteria  which  constitute  their  normal  food. 

Intracellular  digestion  in  the  Infusoria  unquestionably  takes 
place  as  the  result  of  the  action  of  some  diastase ;  but  from  the 
impossibility  of  observing  the  action  in  vitro  the  properties  of  this 
Diastase,  except  that  it  can  act  in  a  distinctly  acid  medium,  cannot  be 
determined. 

Even  less  is  known  concerning  the  digestion  of  Rhizopods  than 
concerning  that  of  Infusoria.  It  has  long  been  recognised  that, 
in  the  majority  of  cases,  Amoeba,  Actmophrys  and  Rhizopods  in 
general,  absorb  a  nourishment  composed  of  lower  plants  and 
animals,  which  are  taken  into  the  protoplasmic  body  by  means  of 
the  movements  of  amoeboid  processes,  pseudopodia  or  lobopodia. 
[17]  Once  within  the  Rhizopod  the  nutritive  particles  are  surrounded 
by  a  digestive  fluid,  in  which  the  presence  of  acid,  may  be  recog- 
nised by  means  of  colour  reactions.  The  addition  of  a  drop  of 
Ehrlich's  neutral-red  to  Amoebae  in  the  act  of  digesting  Bacteria 


Immunity  in  Unicellular  Organisms  15 

at  once  gives  the  acid  colour  reaction  (Fig.  1 ).  Rhumbler1  has  described 

very  precisely  and  with  much  detail  the  way  in  which 

the  Amoebae  behave  when  they  are  incorporating 

filaments  of  Oscillaria  very  much  longer  than 

their  own   bodies.    He  has   also    described   the 

digestion  that  these  Algae  undergo ;   a  process 

most  characteristic  in  those  cases  where  a  portion 

only  of  the  filament  has  been  taken  into  the 

interior  of  the  Amoeba  and  there  subjected  to    Fig    x     An  Amoel)a 

the  digestive    action.     Whilst   the   free  part  of       treated  with  neutrai- 

the  Oscillaria  retains  its  normal  properties  and 

appears  of  a  bluish  green  colour,  the  ingested  portion  progressively 

changes   colour,  assuming  first  a  deep  green  tint,  then  becoming 

light    yellow,    orange    yellow,    brown    and    finally   reddish    brown. 

Simultaneously  the    cellulose   wall    of  the   Alga  begins  to  soften, 

and   the   cells  break  up   into   minute   fragments   which    are   soon 

extruded.    The  food  is  seldom   completely  digested  and  there  is 

always   an  abundant  residual  material  which  is  thrown  out  in  the 

form  of  solid  excreta. 

Although  it  is  fully  recognised  that,  in  the  Rhizopods,  digestion 
goes  on  in  a  medium  distinctly  but  feebly  acid,  and  that  the 
intervention  of  some  soluble  ferment  is  essential,  our  ideas  on  this 
subject  were  very  vague  until  the  publication  of  the  researches  of 
Mouton2,  carried  out  with  great  care  in  the  Pasteur  Institute.  In 
order  that  he  might  obtain  exact  results  Mouton  made  use  of 
cultures  of  Amoebae  grown  on  agar,  in  association  with  the  Bacillus 
coli  which  served  them  as  food.  The  bacilli  were  ingested  in  large 
numbers,  became  enclosed  in  vacuoles  and  were  digested  by  a 
ferment  which  Mouton  was  able  to  obtain  in  vitro.  To  that  end 
he  collected  large  numbers  of  Amoebae,  and,  after  centrifugalising 
them  in  water,  treated  the  deposit  with  glycerine.  On  adding  alcohol  [18] 
he  obtained  a  precipitate  readily  soluble  in  water. 

The  fluid  thus  obtained  exerted  an  undoubted  digestive  action 
upon  albuminoid  substances.  It  readily  liquefied  gelatine  and  even 
attacked,  though  feebly,  albumen  coagulated  by  heat ;  flakes  of  fibrin 
heated  to  58°  C.  remained  unaltered.  There  was  present  then,  in 
this  fluid  derived  from  Amoebae,  a  proteolytic  diastase  of  feeble 
activity.  On  the  other  hand,  this  extract  contained  neither  sucrase, 

1  Arch.f.  Entwickelungsmech.,  Leipzig,  1898,  Bd.  vn. 

2  Compt.  rend.  Acad.  d.  Sci.,  Paris,  1901,  t.  cxxxm,  p.  244. 


16  Chapter  I 

capable  of  inverting  cane  sugar,  nor  lipase,  capable  of  digesting  fatty 
matters. 

The  amoebo-diastase  of  Mouton  must  be  classified  with  the 
trypsins.  It  is  very  active  in  a  distinctly  alkaline  medium  and 
continues  the  diastatic  action  even  when  the  medium  becomes 
weakly  acid  (a  feature  that  corresponds  to  the  reaction  observed 
in  Amoebae  treated  with  appropriate  staining  agents).  The  amoebo- 
diastase  is  affected  at  as  low  a  temperature  as  54°  C.  and  at  60°  C. 
is  rendered  completely  inactive. 

A  question  of  especial  importance  is  that  concerning  the  action 
of  the  amoebo-diastase  upon  Bacteria.  The  numerous  experiments 
of  Mouton  directed  to  the  solution  of  this  point,  and  made  with 
living  Bacillus  coli  communis,  gave  negative  results.  If,  however, 
these  bacilli  were  previously  killed  by  heat  or  by  chloroform, 
they  were  at  once  attacked  by  the  soluble  amoebo-ferment.  Opa- 
lescent emulsions  of  these  dead  bacilli,  incapable  of  undergoing  self- 
digestion  of  any  kind,  became  transparent  after  remaining  for  some 
time  in  contact  with  the  extract  of  Amoebae.  The  amoebo-diastase, 
then,  undoubtedly  digests  dead  bacilli  in  vitro,  whereas  in  the  body 
of  the  Amoebae  the  ingested  bacteria  are  attacked  whilst  still  living. 
As  a  result  of  these  observations  it  must  be  concluded  that  only  a 
fractional  part  of  the  diastase  is  extracted  in  the  solution  prepared 
by  Mouton. 

This  intracellular  digestion  in  the  Protozoa  serves  not  merely  for 
the  nutrition  of  these  organisms,  but  also  as  a  protection  against 
infective  parasites.  The  protoplasm  of  the  Infusoria,  with  its  vacuolar 
secretions,  has  a  general  digestive  action  on  everything  that  comes 
within  its  reach.  If  the  internal  structures,  such  as  the  nuclei  and 
the  pulsatile  vacuoles,  resist  this  process,  it  is  undoubtedly  because 
they  possess  a  power  of  defending  themselves  against  the  attack  of 
the  digestive  secretions.  Thus,  as  brought  out  in  the  beautiful 
[19]  researches  of  Maupas1,  the  macronucleus  of  the  Paramaecia  is,  at 
a  certain  stage  in  the  life  of  the  Infusorian,  completely  digested  by 
the  protoplasm  just  as  is  any  other  nutrient  substance  introduced 
from  outside.  It  must  be  admitted  that  in  this  case  the  nucleus  has 
ceased  to  produce  the  protective  substance  which,  under  ordinary 
conditions,  interferes  with  its  being  digested. 

A  struggle  similar  to  that  observed  between  the  nucleus  and  the 
digestive  content  of  the  Protozoa  goes  on  between  these  latter 

1  Arch,  de  zool.  exptr.,  Paris,  1889,  2me  serie,  t.  vu,  p.  446. 


Immunity  in  Unicellular  Organisms  17 

organisms  and  infective  microbes.  All  organisms  which,  in  any  way 
whatever,  penetrate  into  the  body  of  an  Infusorian  or  Rhizopod,  are 
brought  into  contact  with  the  digestive  endoplasm  of  these  Protozoa. 
If  the  intruders  are  killed  and  partially  oligested  by  the  digestive 
secretions,  or  are  expelled  as  excrementitious  matter,  the  Protozoon 
remains  uninjured  and  continues  its  normal  and  routine  existence. 
Here,  then,  we  have  an  example  of  natural  immunity,  due  to  intra- 
cellular  digestion.  On  the  other  hand,  when  the  foreign  parasitic 
organism  resists  this  digestive  action,  it  instals  itself  permanently  in 
the  body  of  the  Protozoon,  and  should  it  reproduce  itself  in  small 
numbers  merely,  excrete  no  poison  and,  in  general,  exercise  no 
injurious  influence  upon  its  host,  the  parasite  may  readily  become 
a  commensal.  Thus,  it  is  not  rare  to  find  in  the  contents  of  Infusoria 
and  Radiolaria  small  vegetable  organisms  of  the  genera  Zoochlordla 
or  Zooxanthella  which  not  only  set  up  no  disease  but,  owing  to  their 
assimilation  of  carbonic  acid,  may  even  be  useful  to  their  hosts. 
There  are  cases,  however,  where  the  parasites  act  in  a  manner  more 
or  less  injurious  to  the  Protozoa  containing  them ;  in  such  cases 
a  true  and  sometimes  fatal  infection  results. 

Among  the  infective  diseases  of  the  Protozoa  the  one  that  has  been 
most  thoroughly  studied  is  that  set  up  by  several  representatives  of 
a  particular  genus  of  micro-organisms  discovered  by  Johannes  Miiller 
in  1856  and  made  the  subject  of  an  investigation  carried  out  in  my 
laboratory  by  Hafkine1.  I  have  already  discussed  these  researches 
in  my  work  on  the  comparative  pathology  of  inflammation8  and  need 
here  recapitulate  only  very  briefly.  Paramaecia  are  sometimes  affected 
by  needle-shaped  or  spirillar  parasites  which  penetrate,  sometimes 
I  into  the  macronucleus,  sometimes  into  the  micronucleus,  reproducing 
prolifically,  giving  rise  to  a  marked  hypertrophy  of  the  affected  organs. 
The  Infusorian,  in  spite  of  this  invasion,  may  continue  to  exist  and 
carry  on  its  reproductive  processes  ;  it  is,  thus,  enabled  in  many  cases  [20] 
to  recover  from  the  disease.  On  the  other  hand  the  Paramaecium 
into  whose  body  the  spores  of  the  parasite  are  introduced  treats 
I  them  as  it  would  any  other  ingested  foreign  body.  Not  being  able  to 
Ligest  them,  owing  to  the  resistance  offered  by  the  membrane  of  the 
jspore,  the  Paramaecium  expels  them  just  as  it  would  any  other 

1  Ann.de  VInst.  Pasteur,  Paris,  1890,  t.  rv,  p.  148. 

"Lemons  sur  la  pathologic  comparee  de  rinflammation,"  Paris,  1892,  p.  24; 
"Lectures  on  the  comparative  pathology  of  inflammation,"  authorised  translation 
into  English,  London,  1893,  p.  20. 


18  Chapter  I 

excrementitious  matter.    The  Infusorian  behaves  in  the  same  way  in 
regard  to  bacterial  endospores. 

Hay  bacilli,  which  occur  so  commonly  in  the  infusions  in  which 
the  Paramaecia  live,  are  digested  in  the  endoplasmic  vacuoles  of  the 
latter,  but  the  spores  of  these  bacilli,  after  a  more  or  less  prolonged 
sojourn  in  the  vacuoles,  are  expelled  with  the  excrement. 

As  by  far  the  greater  part  of  the  body  of  a  Protozoon  is  made  up 
of  digestive  protoplasm,  it  is  natural  that  infective  epidemics  should 
be  very  rare  among  these  animalcules.  The  Infusoria  and  Rhizopods, 
organisms  specially  well  adapted  to  live  upon  the  lower  Algae  and 
Bacteria,  are,  practically,  never  subject  to  bacterial  diseases.  The 
infections  observed  in  the  Protozoa  are  due  in  most  cases  to  the 
invasion  of  the  lower  Fungi,  such  as  the  Chytridia,  the  Microspheres, 
the  Saprolegniae  or  the  special  organisms  mentioned  as  occurring  in 
the  nuclei  of  Paramaecia.  Further,  these  infections  are  met  with 
most  frequently  in  Protozoa  which  are  incapable  of  carrying  on  true 
intracellular  digestion  or  which  are  in  the  encysted  stage,  at  which 
period  the  Infusoria,  leading  a  passive  existence,  neither  absorb  nor 
digest  nutriment.  As  an  exception  to  the  above  general  statement 
I  ought  to  mention  the  epidemic  in  Amoebae  caused  by  the  Micro- 
sphaera1  and  the  disease  in  Actinophrys  observed  by  K.  Brandt2  and 
attributed  to  Fungi  allied  to  the  genus  Pythium.  In  these  two 
instances  we  have  to  do  with  parasites  which  live  and  develop  in  the 
interior  of  the  active  protoplasm  of  these  Protozoa.  Certainly  a 
proportion  of  the  parasites  are  expelled  with  the  excrementa ;  but 
there  remain  others  which  instal  themselves  in  the  protoplasm, 
multiply  there  and  cause  the  death  of  their  hosts.  In  these  cases  the 
digestive  action  of  the  protoplasm  must  be  neutralised  or  paralysed 
by  the  secretions  of  the  parasite.  This  aspect  of  the  question, 
however,  has  so  far  not  been  considered. 

In  addition  to  intracellular  digestion  and  the  expulsion  of  para- 
sites by  the  excretory  function,  the  resistance  offered  by  Protozoa  to 
infective  diseases  should,  in  part,  be  ascribed  to  their  great  irrita- 
[21]  bility.  Anyone  who  will  watch  the  manoeuvres  of  Amoebae  or  of 
certain  Infusoria  in  the  midst  of  a  rich  microscopic  flora  and  fauna, 
will  at  once  be  struck  by  the  preferences  which  these  Protozoa 
exhibit  in  the  choice  of  their  food.  Amoebae  are  often  seen 
making  search  for  Diatoms  only,  disdaining  all  other  Algae,  or  again 

1  "  Lesons  sur  la  pathologic  comparee  de  1'inflammation,"  p.  21 ;  English  edition,  p.  17. 

2  Monatsler.  d.  Berl  A /cad.  d.  Wissensch.,  1881,  p.  388. 


Immunity  in  Unicellular  Organisms  19 

they  may  single  out  one  species  of  Palmellaceae  from  a  very  varied 
flora.  The  Infusoria  also  have  their  likes  and  dislikes  in  the  matter 
of  food.  Many  of  the  ciliated  Infusoria  choose  Bacteria  to  the  ex- 
clusion of  almost  everything  else ;  others,  as  Nassula,  have  a  special 
partiality  for  the  Oscillariae.  A  most  striking  example  of  this 
is  afforded  in  Amphileptus  claparedei,  a  voracious  Ciliate,  which 
chooses  Vorticellae  to  the  exclusion  of  all  other  animalcules ;  these 
it  devours,  and  then  becomes  transformed  into  a  cyst  upon  tho 
peduncle  of  the  Vorticellae  it  has  devoured.  This  irritability  clearly 
must  control  and  guide  the  Protozoa  in  their  relations  with  other 
organisms  and  enable  them  to  escape  the  invasion  of  parasites. 

In  this  connection  I  must  mention  a  very  interesting  observation 
made  by  Salomonsen1  and  communicated  to  the  Paris  International 
Medical  Congress  in  1900.  He  was  able  to  demonstrate  the  fact  that 
almost  all  the  ciliated  Infusoria,  on  becoming  aware  of  the  proximity 
of  dead  bodies  of  kindred  organisms,  rapidly  draw  away,  thus 
manifesting  a  very  marked  negative  chemio taxis.  This  property 
must,  it  is  evident,  protect  them  from  any  contamination  by  the 
parasites  contained  in  the  bodies  of  Infusoria  that  have  succumbed 
to  infective  diseases. 

We  have,  then,  quite  a  number  of  facts  which  throw  light  on  the 
latural  immunity  of  the  Protozoa  against  the  action  of  pathogenic 
micro-organisms.  Up  to  the  present,  however,  we  know  nothing 
concerning  the  existence  or  the  possibility  of  an  acquired  immunity 
among  the  lower  animalculae  against  infective  diseases.  We  are 
setter  informed  as  to  the  resistance  of  unicellular  organisms  to  the 
action  of  soluble  poisons,  which  is,  in  general,  much  more  easily 
studied  than  is  immunity  against  the  micro-organisms  themselves. 

As  a  very  large  number  of  the  higher  animals  are  sensitive  to  the 
ioxic  action  of  poisons  of  bacterial  origin,  the  question  has  been  put, 
"  May  not  the  Infusoria  also  be  poisoned  by  these  micro-organismal 
products  ?"  With  the  object  of  answering  this  question  Gengou2 
has  studied  the  influence  of  the  toxins  of  tetanus  and  diphtheria  on  [22] 
the  ciliated  Infusoria.  He  was  unable,  however,  to  bring  forward 
proof  that  these  substances  exert  any  special  toxic  action  on  the 
Paramaecia.  These  Infusoria  withstand,  perfectly  well,  doses  of 

1  Compt.  rend,  du  Congres  internat.  de  Med.  tenu  d,  Paris  en  1900.    Section  de 
bacteriologie  et  de  parasitologie. 

2  "Sur  I'immunite  naturelle  des  organismes  monocellulaires  centre  les  toxines" 
Ann.  de  VInst.  Pasteur,  Paris,  1898,  t  xn,  p.  465. 

29 
— & 


20  Chapter  I 

cultures  of  the  diphtheria  and  the  tetanus  bacillus  grown  in  broth 
and  deprived  of  the  bacilli  by  filtration  as  large  as  those  of  ordinary 
broth  alone  in  which  no  bacilli  have  been  cultivated.  Gengou 
argues  from  this  that  the  Paramaecia  possess  a  natural  and  absolute 
immunity  against  these  two  toxins.  When  we  take  into  consideration 
the  fact  that  these  poisons  act  but  feebly  at  ordinary  temperatures 
and  are  often  innocuous  to  "  cold-blooded  "  animals  we  may  perhaps 
be  tempted  to  attribute  the  immunity  of  the  Infusoria  to  the  tem- 
perature that  was  maintained  in  the  incubator  whilst  Gengou's 
experiments  were  being  carried  on.  Led  by  this  train  of  thought 
Mme  MetchnikoiF  tried  the  action  of  the  blood-serum  of  eels,  which 
is  very  toxic,  not  only  for  warm-blooded  Vertebrates  but  also  for  cold- 
blooded Vertebrates  and  the  In  vertebrates,  on  the  Paramaecia,axid  this 
at  a  low  or  medium  temperature.  This  eel's  serum,  however,  exerted 
no  greater  toxic  action  than  did  the  blood-serum  of  other  animals. 

The  microbial  toxins  are  innocuous  not  only  to  the  ciliated 
Infusoria  but  also  to  many  other  unicellular  organisms.  It  is  now 
well  recognised  that  these  toxins,  exposed  to  the  air,  are  soon 
inhabited  by  quite  a  rich  flora  of  micro-organisms,  amongst  which 
Bacteria  and  Yeasts  predominate.  I  have  been  able  to  prove1  that 
these  organisms  are  not  only  unaffected  in  their  normal  life  by  the 
presence  of  the  toxins  of  diphtheria  or  tetanus  but  that  they  rapidly 
bring  about  the  more  or  less  complete  destruction  of  these  poisons. 
Gengou,  also,  observed  that  yeasts  thrive  luxuriantly  in  these  bacterial 
toxins.  The  rapid  increase  of  micro-organisms  and  the  destruction 
of  these  poisons  take  place  at  temperatures  varying  from  15°  to  37°  C. 

Whilst  the  lower  organisms  are  refractory  to  bacterial  toxins 
which  in  quite  small  doses  are  capable  of  killing  man  and  the 
higher  animals,  many  micro-organisms  manifest  a  special  sensitive- 
ness to  certain  fluids  of  animal  origin.  In  a  succeeding  chapter 
we  shall  treat  at  greater  length  of  this  microbicidal  property  of 
the  humours.  Here  it  is  merely  necessary  to  indicate  certain  facts 
concerning  this  property,  regarding  them  solely  from  the  point  of 
[23]  view  of  the  immunity  of  the  lower  organisms.  The  most  striking 
example  of  the  bactericidal  power  of  an  animal  fluid  is  certainly 
that  afforded  in  the  action  of  the  blood-serum  of  the  rat  on  the 
anthrax  bacillus.  This  fact,  discovered  in  1888  by  von  Behring2,  led 

1  Ann.  de  VInst.  Pasteur,  Paris,  1897,  t.  xi,  p.  801. 

2  "Ueber  die  Ursache  der  Immunitat  von  Ratten  gegen  Milzbrand,"  in  the 
Centralbl.f.  klin.  Med.t  Bonn,  1888,  no.  38. 


Immunity  in  Unicellular  Organisms  21 

to  the  conclusion  that  the  blood  of  the  rat  contains  an  organic  base 
capable  of  killing  and  dissolving  a  considerable  number  of  anthrax 
bacilli  Several  observers  have  confirmed  von  Bearing's  observation 
and  have  supplemented  it  by  the  fact  that  the  bacillus  can  be  readily 
accustomed  to  the  toxic  action  of  this  serum.  Thus  Sawtchenko1,  in 
an  investigation  carried  out  in  my  laboratory,  was  able,  by  successive 
cultures,  to  accustom  the  anthrax  bacillus  to  an  existence  in  the  pure 
serum  of  the  rat.  In  this  case,  therefore,  there  has  been  produced  a 
real  acquired  immunity  of  a  lower  plant  against  a  toxic  substance  of 
animal  origin.  More  recently  Danysz  has  demonstrated  the  same  thing 
and  has  added  several  other  facts  which  seem  to  throw  light  upon  the 
means  by  which  the  bacterium  becomes  adapted  to  the  poison.  He 
has  shown,  in  a  work  carried  out  in  the  Pasteur  Institute 2,  that  the 
anthrax  bacillus  protects  itself  against  the  toxic  action  of  the  serum 
by  surrounding  itself  with  a  thick  sheath  composed  of  a  kind  of 
mucus  which  fixes  the  toxin  of  the  rat's  blood  and  renders  it 
harmless.  This  same  mucus,  but  in  smaller  quantity,  is  likewise 
produced  in  a  culture  of  the  bacillus  grown  in  ordinary  broth.  When 
such  a  culture  is  freed  from  the  contained  bacilli  by  filtration  through 
porcelain  and  a  little  of  this  fluid  is  added  to  the  rat's  serum,  this 
latter  becomes  less  bactericidal  than  is  a  mixture  of  the  same  serum 
with  ordinary  broth.  Danysz  suggests  that  this  is  to  be  explained 
by  the  presence  in  the  filtrate  of  a  certain  quantity  of  the  mucous 
substance  produced  by  the  bacillus,  which  fixes  and  neutralises  a 
portion  of  the  "rat  toxin."  If,  in  place  of  sowing  the  ordinary 
bacillus,  sensitive  to  this  toxin,  we  inoculate  the  broth  with  an 
anthrax  bacillus  which  has  previously  been  accustomed  to  the  rat's 
serum,  we  find  that  the  liquid  of  this  culture  when  filtered  neutralises 
a  larger  proportion  of  the  toxin.  Danysz  concludes  from  this  that  the 
acclimatised  bacillus  has  acquired  the  property  of  producing  more 
mucus  than  does  the  ordinary  bacillus  and  that,  for  this  reason,  a 
greater  quantity  of  this  protective  substance  passes  into  the  fluid  of 
the  culture. 

The  formation  of  a  transparent  sheath  has  several  times  been  [24] 
observed  in  the  anthrax  bacillus,  notably  in  cases  where  this  organism 
happens  to  be  in  "a  state  of  defence"  against  various  noxious 
influences.      For  example,   this  sheath    is  well    developed   in   the 
anthrax  bacillus  which  invades  the  blood  of  lizards,  animals  which 

1  Ann.  de  VInst.  Pasteur,  Paris,  1897,  t.  xi,  p.  872. 

2  Ann.de  Flnst.  Pasteur,  Paris,  1900,  t.  xiv,  p.  641. 


22 


Chapter  I 


are  in  general  very  resistant  to  anthrax1.  Under  analogous  conditions 
the  streptococci  which,  as  a  rule,  do  not  produce  a  mucous  sheath, 
will  develop  one  of  exceptional  size.  The  guinea-pig  is  in  general 
very  resistant  to  the  streptococcus  against  which  it  exhibits  a  very 
effective  reaction.  Sometimes,  however,  this  immunity  gives  way  ; 
in  such  instances,  as  demonstrated  by  J.  Bordet2,  the  streptococcus,  in 
order  to  overcome  the  natural  resistance  of  the  guinea-pig,  is  found 
to  have  surrounded  itself  with  a  sheath  of  a  thickness  such  as  is 
seldom  to  be  met  with  in  the  world  of  bacteria  (Fig.  2). 


Fig.  2.     Streptococcus  surrounded 
by  a  protective  envelope. 


Fig.  3.  Tubercle  bacillus  surrounded 
by  a  transparent  envelope  and  en- 
closed in  the  giant  cell  oi  a  gerbil. 


Analogous  facts  are  also  observed  in  cases  where  the  micro- 
[25]  organism  is  defending  itself  against  the  action  of  substances  enclosed 
in  animal  cells.  I  may  cite  as  an  example  the  tubercle  bacillus  in 
the  interior  of  the  giant  cells  of  a  gerbil  (Meriones  shawii),  where, 
under  the  influence  of  noxious  substances  contained  in  these  cells,  the 
tubercle  bacillus  (Fig.  3)  envelops  itself  in  a  transparent  sheath 
similar  to  that  of  the  bacillus  or  of  the  streptococcus.  As  the  action 
of  the  giant  cell  still  does  not  cease,  the  tubercle  bacillus  secretes 
a  second  sheath  (Fig.  4)  and  continues  to  surround  itself  with 

1  Metchnikoff,  Virchow's  Archiv,  1884,  Bd.  xcvn,  S.  510. 

2  "  Contribution  a  1'etude  du  serum  antistreptococcique,"  Ann.  de  I'Inst.  Pasteur, 
Paris,  1897,  t.  xi,  p.  177,  Planche  V. 


Immunity  in  Unicellular  Organisms  23 

quite  a  series  of  such  envelopes  (Fig.  5),  thus  coming  to  resemble 
a  palmellaceous  Alga  surrounded  by  successive  layers  of  membranes 
or  certain  other  vegetable  cells  whose  principal  means  of  defence 
against  all  kinds  of  injurious  influences  consists  in  the  production 
of  these  protective  membranes. 


Fig.  4.     Another  tubercle  bacillus  Fig.  5.     Tubercle  bacillus  surrounded 

surrounded  by  two  membranes.  by  a  series  of  concentric  layers. 

Quite  recently  Trommsdorf1,  in  Buchner's  laboratory  in  Munich, 
has  carried  out  a  series  of  experiments  on  the  adaptation  of 
the  cholera  vibrio  and  of  the  typhoid  bacillus  to  the  bactericidal 
substance  found  in  the  blood  of  the  rabbit.  He  has  been  able  to 
confirm  the  results  of  his  predecessors  and  by  various  experiments 
has  convinced  himself  that  these  two  micro-organisms  are  capable 
of  adapting  themselves  to  existence  in  the  defibrinated  blood  and  in  [26] 
the  blood-serum  of  the  rabbit. 

The  immunity,  or  acclimatisation  of  injurious  organisms  to  different 
toxins,  presents  an  undoubted  analogy  to  the  phenomena  of  adaptation 
shown  by  these  organisms  to  mineral  or  organic  poisons.  It  has  long 
been  known  that  the  same  species  of  Protozoa  are  met  with  in  both 
fresh  and  salt  water  and  that  it  is  possible  to  gradually  accustom 
Infusoria  and  Amoebae  to  tolerate  an  amount  of  sea  salt  which  at  first 
is  absolutely  fatal  to  them.  This  toleration  is  not  acquired  unless  care 
be  taken  to  increase  the  amount  of  salt  very  gradually :  too  abrupt  a 
rise  inevitably  causing  death.  By  this  means  Cohn'2  accustomed  the 

1  Arch.f.  Hyg.,  Miinchen  u.  Leipzig,  1900,  Bd.  xxxix,  S.  31. 

2  "  Entwickelungsgeschichte  der  mikroskopischen  Algen  und  Pilze,"  Nov.  Acta 
Acad.  Goes.  Leap.  Carol.,  1854,  t.  xxiv,  p.  1. 


24  Chapter  I 

fresh- water  Euplotes  to  a  life  in  artificial  sea  water  containing  4  °/0  of 
sodium  chloride.  In  Balbiani's  experiments '  the  fresh-water  Monads 
(Menoidium  incurvum  and  Chilomonas  paramaecium)  died  very 
quickly  on  the  addition  of  J  %  of  this  salt ;  but  when  it  was  added  in 
small  successive  doses  (0'05  per  day),  they  readily  became  accustomed 
to  a  concentration  of  1  %•  In  the  encysted  state  the  Protozoa  are  even 
more  resistant  than  in  the  active  state  to  the  different  salts  that  may 
be  added  to  their  normal  culture  medium.  It  is  probable  that  the 
wall  of  the  cyst  interferes  with  the  penetration  of  these  substances 
into  the  endoplasm.  If  a  small  quantity  of  an  aniline  dye  be  added  to 
a  fluid  containing  encysted  Infusoria,  it  is  seen  that  the  cyst-membrane 
becomes  very  intensely  coloured  but  the  body  of  the  Infusorian 
remains  unstained.  The  membrane  absorbs  a  large  amount  of 
colouring  matter,  after  which,  being  saturated,  it  ceases  to  take  it 
up  ;  but  it  does  not  allow  the  dye  to  penetrate  into  the  endoplasm. 

Balbiani  (loc.  dt.  p.  580),  having  compared  the  action  of  the  salts 
of  sodium  with  that  of  the  salts  of  potassium  and  lithium  on  In- 
fusoria, comes  to  the  conclusion  that  the  injurious  influence  of  these 
substances  can  only  be  partially  explained  by  osmotic  phenomena. 
In  addition  to  these  a  purely  chemical  action  must  be  invoked.  He 
bases  his  opinion  on  the  fact  that  the  isotonic  solutions  of  the  three 
[27]  salts  acting  on  Infusoria  of  the  same  species  and  same  origin  exert 
a  different  influence.  The  salts  of  potassium  and  of  lithium  act  in  a 
much  more  energetic  fashion  than  do  the  sodium  salts.  Consequently, 
the  Protozoa  are  able  to  adapt  themselves  progressively  not  only  to 
noxious  influences  of  a  physiological  character  but  also  to  those  of  a 
chemical  nature.  Thus  Infusoria  and  Rhizopods  can  be  accustomed  to 
the  action  of  high  temperatures,  to  an  intense  light,  etc.  On  the  other 
hand  they  can  also  be  habituated  to  the  toxic  actions  of  true  poisons. 
Davenport  and  Neal2  have  established  the  fact  that  Stentors  kept  for 
two  days  in  a  weak  solution  of  corrosive  sublimate  (0-00005  %)  acquire 
a  tolerance  to  a  dose  of  this  poison  four  times  as  great  as  the  lethal 
dose  for  individuals  previously  kept  in  pure  water.  The  same  thing 
has  been  observed  in  connection  with  the  toxic  action  of  quinine. 
This  immunity  cannot  be  attributed  to  the  selection  and  persistence 
of  those  Infusoria  which  possess  a  natural  resistance  to  the  sublimate. 

1  "Action  des  sels  sur  les  infusoires,"  Arch,  d-anat.  microsc.,  Paris,  1898,  t  n, 
p.  595. 

2  "  On  the  acclimatisation  of  organisms  to  poisonous  chemical  substances,"  Arch, 
f.  Entwickelungsmech.,  Leipzig,  1895,  Bd.  n,  S.  564. 


Immunity  in  Unicellular  Organisms  25 

It  is  really  acquired  as  the  result  of  a  direct  and  gradual  chemical 
influence  on  the  protoplasm  of  the  Stentors  which,  once  adapted, 
all  survive  doses  which  are  lethal  for  the  unacclimatised  control 
organisms. 

The  vegetable  micro-organisms,  which  are  much  more  easily 
cultivated  than  are  the  Protozoa,  frequently  manifest  most  charac- 
teristic phenomena  of  acclimatisation.  The  first  systematic  researches 
in  this  direction  were  carried  out  by  Kossiakoff l  in  the  laboratory  of 
Duclaux.  He  studied  the  antiseptic  action  of  borax,  of  boracic  acid, 
and  of  corrosive  sublimate  on  the  anthrax  microbe  and  several  other 
bacilli  (Bacillus  subtilis,  Thyrotrix  scaber  and  T.  tennis).  He  found 
that  all  these  micro-organisms  can  be  gradually  accustomed  to  doses 
which  are  absolutely  bactericidal  to  the  same  species  when  not  so 
acclimatised.  The  acclimatised  Thyrotrix  tennis  withstands  almost 
double  the  amount  of  bichloride  of  mercury  that  the  non-acclimatised 
bacillus  will  resist.  The  ordinary  anthrax  bacillus  will  not  develop  at  all 
if  the  culture  medium  contains  more  than  0*005  of  boracic  acid  whilst 
the  same  organism,  when  accustomed  by  passage  through  successive 
cultures  in  which  this  substance  is  present  in  gradually  increasing 
proportions,  grows  well  in  spite  of  the  presence  of  0'007  of  the  same 
antiseptic.  Since  these  observations  were  made  similar  facts  have 
been  demonstrated  by  several  other  observers,  and  the  ready  accli- 
matisation of  Bacteria  to  poisons  is  now  generally  admitted.  Danysz  [28] 
(loc.  cit.\  with  the  object  of  elucidating  the  mechanism  of  this  adapta- 
tion, has  studied  the  action  of  arsenic  acid  on  the  Bacillus  anthracis. 
He  has  demonstrated  that  this  bacillus  will  gradually  accustom 
itself  to  grow  in  broth  containing  a  quantity  of  arsenic  acid  which  at 
first  inhibited  all  development.  During  this  phenomenon  of  adapta- 
tion, which  is  acquired  after  a  series  of  passages  through  media  more 
and  more  highly  arsenicated,  the  bacillus  secretes  a  coating  of  mucous 
substance  which  protects  the  sensitive  parts  of  the  microbial  cell. 
Here,  therefore,  is  formed  something  exactly  corresponding  to  what 
the  same  observer  has  demonstrated  in  anthrax  bacilli  that  have 
acquired  a  tolerance  for  rat's  serum.  This  analogy  extends  even 
to  the  throwing  out  of  the  protective  substance  into  the  culture 
fluid.  When  one  sows  an  ordinary  unadapted  bacillus  in  arsenicated 
broth  to  which  has  been  added  some  of  the  fluid  from  a  culture  of 
the  adapted  bacillus,  development  takes  place  in  a  marked  fashion. 
On  the  contrary  when  the  same  material  is  "  seeded  "  into  arsenicated 
1  Ann.  de  VInst.  Pasteur,  Paris,  1887, 1. 1,  p.  465. 


26  Chapter  1 

broth  of  the  same  composition  but  to  which  has  been  added  the 
filtrate  from  an  unadapted  culture,  the  bacillus  does  not  develop 
nearly  so  well.  The  difference  is  explained  by  the  presence,  in  the 
fluid  in  which  the  adapted  bacillus  had  grown,  of  a  certain  quantity 
of  the  mucous  substance  which  fixes  the  arsenic  and  prevents  it  from 
acting  on  the  protoplasm  of  the  micro-organisms. 

The  Yeasts,  also,  adapt  themselves  very  readily  to  antiseptics. 
This  property  has  even  had  a  practical  application.  We  know  that 
small  doses  of  hydrofluoric  acid  are  capable  of  preventing  the 
proliferation  of  the  yeast  of  beer,  and  Effront1  has  accustomed  this 
plant  to  live  in  media  containing  an  amount  of  hydrofluoric  acid 
which  is  absolutely  inhibitory  to  the  unadapted  yeast.  Under  these 
conditions  the  adapted  cells  undergo  a  stimulation  which  causes  the 
production  of  a  greater  quantity  of  alcohol.  The  yeast,  in  adapting 
itself  to  antiseptic  doses  (300  mm.  of  hydrofluoric  acid  per  100  c.c.  of 
beer  wort),  acquires  a  kind  of  immunity  which  it  did  not  possess  in 
the  first  instance.  Moreover  this  acquired  property  can  be  hereditarily 
transmitted  to  new  generations  developed  in  ordinary  beer  wort  to 
which  hydrofluoric  acid  has  not  been  added.  The  stimulating  action 
of  this  substance  on  the  fermentative  property  does  not  depend  upon 
the  acid  reaction  of  the  hydrofluoric  acid,  for  other  acids  which  are 
[29]  non-antiseptic,  such  as  tartaric  acid,  are  incapable  of  inducing  it. 

The  acquired  immunity  against  hydrofluoric  acid  is  strictly  specific, 
the  yeasts  that  have  been  adapted  to  this  substance  becoming  even 
more  susceptible  to  the  action  of  other  poisons. 

Duclaux2  has  already  insisted  on  the  relations  which  exist  between 
antiseptics  and  foods.  Formic  aldehyde  which  has  a  very  powerful 
coagulative  and  therefore  strongly  antiseptic  action  on  protoplasm  may 
actually  serve  as  a  food  for  micro-organisms.  The  Thyrotrix  tennis, 
studied  in  this  connection  by  Pere* 3,  adapts  itself  to  the  presence  of  this 
aldehyde  and  utilises  it  for  its  nutrition.  Here  is  produced  something 
that  recalls  the  case  of  the  Protozoa  that  digest  parasitic  organisms. 

It  is  now  a  current  idea  in  microbiology  that  Bacteria  and 
Yeasts  which  primarily  do  not  make  use  of  certain  substances,  adapt 
themselves  to  use  them  as  nutrient  substances.  Dienert4  has  published 
a  detailed  work  on  the  adaptation  of  the  yeasts  to  milk-sugar.  This 

1  Monit.  stient.  du  Dr  Quesneville,  1890,  1891,  1892,  1894. 

2  "Traite  de  Microbiologie,"  Paris,  1898,  t.  I,  p.  238. 

3  Ann.  de  TInst.  Pasteur,  Paris,  1896,  t.  x,  p.  417. 

4  Ann.  de  VInst.  Pasteur,  Paris,  1900,  t.  xiv,  p.  139. 


Immunity  in  Unicellular  Organisms  27 

sugar  is  usually  disdained  by  the  yeasts  that  set  up  the  fermentation 
of  glucose ;  it  is  not  difficult,  however,  to  adapt  them  to  galactose 
which  they  then  attack  and  transform  into  alcohol  and  carbonic  acid. 

The  Protozoa  can  be  progressively  accustomed  not  only  to  poisons 
but  also  to  altered  physical  conditions.  Thus,  Dallinger1  succeeded 
in  raising  the  temperature  of  the  water  in  which  flagellated  Infusoria 
were  growing  from  15°*5  to  23°  C.  without  causing  their  death.  By 
prolonging  the  experiment  over  several  months,  he  was  even  able  to 
habituate  them  to  an  existence  at  a  temperature  of  70°  C.  In  the 
opinion  of  Davenport2,  a  view  which  is  shared  by  many  other 
observers,  this  resistance  to  high  temperatures  was  dependent  on 
the  abstraction  of  water  from  the  protoplasm.  Dallinger  has  also 
observed  that  in  Infusoria  that  are  accustomed  to  life  in  hot  water, 
the  vacuoles  become  smaller  and  smaller  and  may  even  actually 
disappear. 

This  adaptation,  then,  is  a  property  that  is  very  general  and  wide- 
spread in  the  microcosm  of  the  unicellular  organisms.  It  is  connected 
with  the  intracellular  digestion  of  solid  food  and  with  the  absorption 
and  transformation  of  soluble  substances.  These  phenomena,  chemical 
in  character,  are  intimately  linked  with  the  irritability  of  microscopic 
organisms,  which  represents  one  of  the  fundamental  properties  of  [30] 
living  organisms. 

A  Protozoon,  which  is  refractory  to  a  parasite,  may  protect  itself 
by  flight  or  it  may  devour  and  digest  the  parasite ;  another,  which 
acquires  a  tolerance  in  regard  to  a  toxin  or  to  a  mineral  poison, 
absorbs,  fixes  and  transforms  this  substance.  Consequently,  in  all 
these  instances  of  immunity  there  is  a  reaction  of  the  living  elements 
of  the  organism,  this  being  a  direct  consequence  of  the  irritability  of 
the  protoplasm. 

Before  an  Infusorian  retreats  from  the  dead  body  of  an  allied 
organism,  before  a  Protozoon  secretes  a  digestive  fluid  around  the 
prey  it  has  ingested,  before  a  Bacterium  secretes  a  glairy  layer  for 
its  defence,  etc.,  these  unicellular  organisms  must  receive  sensations 
which  provoke  the  above-mentioned  reactions.  It  is  to  a  celebrated 
botanist,  Pfeffer,  that  we  owe  the  most  important  researches  on  this 
irritability  of  unicellular  organisms,  researches  which  may  be  summed 
up  in  the  general  statement  that  this  property  is  subject  to  the  psycho- 
physical  law  of  Weber-Fechner.  Pfefler,  by  the  observation  of  the 

1  Journ.  R.  Micr.  Soc.,  London,  1880,  m,  p.  1. 

2  Davenport  and  Castle,  Arch.f.  Entwickelungsmech.,  Leipzig,  1895,  Bd.  n,  S.  227. 


28  Chapter  I 

movements  of  Bacteria  under  the  influence  of  increasing  stimulations, 
has  established  the  fact  that,  conformably  to  this  law,  when  the 
stimulus  increases  in  geometrical  ratio,  the  irritability  increases  in 
arithmetical  ratio,  that  is  to  say,  the  reaction  is  proportional  to 
the  logarithm  of  the  stimulation.  In  order  that  a  motile  bacterium 
(Bacterium  termo),  grown  in  a  peptonised  solution,  may  perceive  a 
difference  of  medium,  it  is  necessary  to  place  it  in  a  peptone  solution 
of  five  times  the  original  concentration ;  weaker  solutions,  in  which  the 
concentration  is  but  three  or  four  times  greater  than  the  original 
fluid,  do  not  attract  the  bacteria  at  all ;  consequently  these  differ- 
ences are  below  their  chemiotactic  sensibility. 

The  different  reactions  that  are  exhibited  in  the  immunity  of 
unicellular  organisms,  reactions  which  are  dependent  on  the  irrita- 
bility of  their  protoplasm,  therefore,  come  undeniably  under  the 
category  of  purely  cellular  phenomena. 


CHAPTER  II  [si] 

IMMUNITY  IN  MULTICELLULAR  PLANTS 

Infective  diseases  of  plants. — Plasmodia  of  the  Myxomycetes  and  their  chemiotaxis. — 
Adaptation  of  the  plasm odia  to  poisons. — Pathogenic  action  of  Sderotinia  upon 
Phanerogams. — The  cicatrisation  of  plants. — Defence  in  plants  against  Bacteria. — 
Sensitiveness  of  vegetable  cells  to  osmotic  pressure. — Adaptation  of  plants  to 
modifications  of  osmotic  pressure. — Dependence  of  the  chemical  phenomena 
upon  the  irritability  of  the  vegetable  cells.— The  law  of  Weber-Fechner. 

FOR  several  reasons  this  immunity  in  the  vegetable  kingdom  cannot 
be  treated  in  a  satisfactory  fashion.  Much  attention  has  been  devoted 
to  the  pathology  of  plants  and  the  etiology  of  a  number  of  vegetable 
diseases  was  well  established  at  a  period  when  we  were  still  groping 
in  the  dark  for  the  causes  of  infective  diseases  in  man  and  the  higher 
animals.  In  spite  of  this,  the  botanist  has  relegated  the  study  of  the 
phenomena  of  immunity  to  a  secondary  position,  and  up  to  the  present 
no  work  specially  devoted  to  this  subject  has  appeared.  It  is  only 
incidentally  that  the  question  of  the  resistance  of  certain  plants  to 
morbific  factors  capable  of  infecting  or  intoxicating  them  has  been 
touched  upon.  We  should  require,  therefore,  to  carry  out  special 
researches  in  this  direction  and  to  make  a  very  complete  study  of 
botanical  literature,  before  we  should  be  able  to  present  to  our 
readers  a  resume  of  the  question  of  immunity  in  the  vegetable 
kingdom.  Such  a  programme  being  impossible  we  must  content 
ourselves  with  borrowing  from  the  botanists  certain  facts  which 
throw  light  on  some  aspects  of  the  general  problem  in  which  we 
are  interested. 

Many  of  the  higher  plants  are  subject  to  infective  diseases  set 
up  by  the  lower  plants,  of  which  the  most  important  are  the  Fungi. 
Whereas  in  the  animal  kingdom  the  majority  of  the  infections 
are  due  to  Bacteria,  these  micro-organisms  rarely  occur  in  plants  ;  [32] 
moreover  when  they  are  present  the  part  they  play  is  nearly  always 
a  secondary  one.  This  difference  is  due  mainly  to  the  chemical 


30  Chapter  II 

composition  of  the  "  humours  "  in  the  two  kingdoms,  the  cell-juice  of 
plants  being  generally  acid  ;  under  this  condition  the  Fungi  develop 
much  better  than  do  the  Bacteria. 

The  various  modes  of  defence  against  infective  diseases  that  have 
been  met  with  in  unicellular  organisms  are  also  found  in  the  multi- 
cellular  plants.  Whereas  in  almost  all  plants  the  cells  are  rigid, 
owing  to  the  presence  of  a  well-developed  membrane,  some  of  the 
lower  plants  have  preserved  a  condition  in  which  the  protoplasm  is 
completely  naked  and  capable  of  movement.  Myxomycetes  are 
specially  distinguished  by  an  amoeboid  stage  of  existence  and  by  the 
formation  of  large  plasmodia  which  protrude  protoplasmic  processes 
and  exhibit  a  kind  of  locomotion  similar  to  that  met  with  in  the 
Rhizopods  and  the  Sporozoa. 

Infective  diseases  among  the  Myxomycetes  must  be  very  rare 
since,  up  to  the  present,  they  have  not  been  noted  by  a  single  observer. 
It  is  very  probable  that  the  plasmodia  get  rid  of  the  infective  germs, 
as  do  the  Protozoa,  both  by  expulsion  of  the  parasites  and  by 
means  of  intracellular  digestion.  This  latter  takes  place  in  a  medium 
which  is  distinctly  acid  and  by  means  of  a  soluble  ferment  described 
by  Krukenberg1  as  a  kind  of  pepsin.  I  need  not  here  enter  into 
further  detail  as  I  have  already  treated  this  subject  in  my  Lectures 
on  the  comparative  pathology  of  inflammation.  The  fact  that  the 
Myxomycetes  can  ingest  living  organisms  has  been  demonstrated  by 
Celakovsky,  jun.a,  who  has  observed  that  the  spores  of  the  various 
Fungi  can  germinate  in  the  interior  of  the  plasmodium.  Whilst  our 
conceptions  concerning  the  resistance  of  the  plasmodia  in  regard  to 
micro-organisms  are  merely  based  upon  analogies  and  hypotheses,  our 
ideas  as  to  their  immunity  against  soluble  substances  rest  on  well- 
established  experimental  facts.  We  owe  to  Stahl3  our  first  informa- 
tion as  to  the  mode  by  which  the  plasmodia  resist  poisons.  When 
they  are  placed  in  contact  with  solutions  of  salts,  of  acids  or  of  sugar 
in  a  sufficiently  concentrated  form  to  bring  about  an  injurious  action, 
the  plasmodia  make  use  of  their  amoeboid  power  of  motion  to  escape 
[ssjfrom  these  fluids.  Hence  they  exhibit  a  negative  chemiotaxis,  ex- 
actly parallel  to  that  so  often  observed  in  the  case  of  the  unicellular 
organisms.  Consequently  there  is  in  the  Myxomycetes  a  natural 
immunity  due  to  the  activity  of  their  movements.  Further,  a  kind 

1  Untersuch.  a.  d.  physiolog.  Inst.  d.  Univ.  Heidelberg,  1878,  Bd.  n,  S.  273. 

2  Flora,  Marburg,  1892,  Bd.  LXXVI,  S.  182. 

3  Boian.  Ztg.,  Leipzig,  1884,  S.  161. 


Immunity  in  Multicellidar  Plants  81 

of  acquired  immunity  in  these  plants  has  also  been  demonstrated 
by  Stahl.  The  following  is  the  passage  in  his  paper  referring  to 
this  subject,  a  passage  very  important  from  a  general  point  of  view1 : 
"  If  we  replace  the  water  in  a  vessel  by  a  1  or  2  %  solution  of 
glucose,  we  observe  either  the  death  of  the  plasmodia,  if  the  action 
is  too  rapid,  or  merely  their  retreat  from  the  glucose  solution. 
Even  solutions  of  ^  or  J%  are  at  first  avoided  by  the  plasmodia 
and,  should  the  action  be  too  rapid,  may  cause  their  death.  Usually 
the  plasmodia  emigrate  into  those  portions  of  the  substratum  remote 
from  the  solution,  to  return  after  some  time,  often  only  after  several 
days,  and  immerse  themselves  in  the  solution  of  glucose  as  they  do 
in  an  infusion  of  tan,  although  with  more  hesitancy.  Consequently 
the  Myxomycetes  accommodate  themselves  slowly*  to  a  more  con- 
centrated solution,  probably  by  giving  up  a  certain  proportion  of  their 
water.  I  was  able  to  observe  the  same  phenomena  with  even  much 
more  concentrated  solutions  (2  %)•  A  plasmodium  which  at  the  end 
of  several  days  had  adapted  itself  to  a  2  %  solution  of  glucose  and  had 
sent  out  numerous  processes  into  it,  found  itself  injuriously  affected 
when  the  sugar  solution  was  suddenly  replaced  by  pure  water.  Those 
that  remained  alive  had  retired  to  a  great  distance  from  the  upper 
layer  of  the  fluid  and  did  not  descend  again  until  the  end  of  the  second 
day.  After  a  fresh  change  of  fluid  we  were  able  to  observe  first  the 
repulsion  and  later  the  attraction  of  the  plasmodia,  but  a  certain  time 
always  elapses  before  the  plasmodia  become  accustomed  to  the  change 
in  concentration.  We  obtain  the  same  result  when  we  replace  a  2  % 
solution,  not  by  pure  water,  but  by  a  ^  or  a  1  °/o  solution  "  (p.  166). 

De  Bary3  had  already  interpreted  these  facts  as  being  due  to  an 
immunity  acquired  by  the  plasmodia,  the  result  of  an  adaptation  of 
these  organisms  to  solutions  which  they  had,  at  first,  carefully  avoided. 
He  threw  out  the  suggestion  that  a  similar  adaptation  might  take[3£] 
place  in  regard  to  solid  substances  ingested  by  the  Myxomycetes. 

As  these  phenomena  of  acquired  immunity,  in  organisms  so 
primitive  and  of  so  simple  a  structure,  are  of  immense  importance 
from  the  point  of  view  of  Immunity  in  general  I  felt  bound  to  submit 
them  to  a  personal  investigation.  I  found  it  an  easy  matter  to 

1  [Stahl  used  plasmodia  which  had  spread  themselves  on  a  substratum  of  wet 
filter  paper  applied  to  the  inside  of  glass  vessels,  its  lower  edge  touching  the  surface 
of  the  experimental  fluid  at  the  bottom  of  the  vessel  (Translator).] 

2  The  italics  are  M.  Metchnikoff's. 

3  "  Vergleichende  Morphologic  u.  Biologic  der  Pilze,  Mycetozoen  u.  Bacterien," 
Leipzig,  lte  Aufl.,  1884  ;  also  authorised  English  translation,  Oxford,  1887. 


32  Chapter  II 

accustom  the  plasmodia  of  Physarum  to  solutions  of  arsenious  acid 
which  at  first  repelled  them  in  a  very  striking  manner.  This  adapta- 
tion manifests  itself  by  movements  of  the  plasmodia  and  by  the 
change  from  negative  chemiotaxis  (repulsion)  to  positive  chemiotaxis 
(attraction). 

It  is  impossible  in  the  present  state  of  our  knowledge  to  state 
precisely  the  modifications  that  the  plasmodia  undergo  during  this 
process  of  adaptation.  Stahl  supposes  that  they  depend  "on  some 
special  properties  of  the  plasmodia  (probably  in  a  greater  or  less 
richness  in  water)";  and  that  it  is  a  case  "not  of  simple  phenomena, 
easy  of  explanation,  but  of  extremely  complicated  phenomena  of 
irritability." 

It  is  evident  that,  in  this  case  of  acquired  immunity,  we  have 
not  to  do  with  a  question  of  physical  or  chemical  modification  of  the 
solutions  employed  but  solely  with  reactive  phenomena  on  the  part 
of  the  living  plasmodia. 

After  a  phase  of  active  life,  during  which  the  Myxomycetes  move, 
feed,  digest  and  expel  waste  products  as  do  the  lower  animals, 
there  comes  a  stage  when  they  become  immobile  and  transform  them- 
selves into  a  number  of  sporangia  filled  with  rounded  spores.  Before 
leaving  their  animal  aspect  for  that  of  true  plants,  the  plasmodia 
exhibit  entirely  new  attributes.  They  reject  all  nourishment  and 
no  longer  ingest  foreign  bodies ;  they  avoid  the  moisture  which 
previously  attracted  them  and  cease  to  shrink  from  the  light. 

Having  come  to  maturity,  the  Myxomycetes  declare  themselves 
true  plants  and  lead  a  passive  life  until  the  new  generation  comes 
forth.  Most  plants  are  restricted  to  this  passive  phase  of  the 
Myxomycetes.  In  these  latter  it  persists  only  for  a  short  period, 
whereas  in  almost  all  plants  it  is  the  permanent  condition.  It 
is  at  this  stage  that  these  organisms  are  liable  to  the  attack  of 
parasites  against  which  it  is  necessary  for  them  to  oppose  all  their 
means  of  defence.  Our  knowledge  of  these  means  of  defence  is  as 
[35]  yet,  as  I  have  already  stated,  very  imperfect,  and  the  example  of 
Sclerotinia  libertiana  (or  Peziza  sclerotiorum)  which  has  been  the 
subject  of  the  researches  of  de  Bary1  remains  up  to  the  present  the 
one  that  has  been  most  thoroughly  studied. 

This  Fungus,  belonging  to  the  group  of  the  Discomycetes,  invades 
many  species  of  plants  and  often  produces  great  .ravages  amongst 
the  cultivated  plants  of  our  fields  and  gardens,  such  as  colza,  hemp 
1  Botan.  Ztg.,  Leipzig,  1886,  SS.  377,  393,  409,  433,  449,  465. 


Immunity  in  Multicellular  Plants  33 

petunias,  dahlias,  etc.  The  mycelium  of  this  Sderotinia  develops  in 
the  stems  of  herbaceous  plants  and  produces  sclerotia  inside  them, 
forms  of  resistance,  which  in  this  instance  are  black  and  resemble 
small  particles  of  mouse  excrement. 

The  spores  of  the  Sderotinia  germinate  and  form  mycelial  threads 
[on  the  surface  of  the  plants.    In  order  that  they  may  penetrate  into 
the  tissues  these  threads  must  attack  the  cell-membrane  and  for  this 
purpose  they  secrete  a  fluid,  which  contains  both  a  digestive  ferment 
nd  oxalic  acid,  the  latter  being  essential  for  the  action  of  the  ferment 
The  presence  of  this  "  toxin  "  has  been  demonstrated  by  de  Bary 
»y  macerating  the  mycelium  of  the  Sderotinia.    The  resultant  extract 
las  a  well-marked  action  on  the  tissues  of  many  plants   (carrot, 
erusalem  artichoke,  chicory,  etc.).    Under  its  influence  the  proto- 
ilasm  of  the  cells  contracts,  a  genuine  plasmolysis  is  set  up,  the  cell- 
embrane  swells  and  its  layers  between  the  cells  are  dissolved.    As 
he  result  of  this  digestive  action,  the  cells  become  separated  and 
he  tissue  softens.     This  extract,  when  heated  to  52°  C.,  loses  its 
igestive  action  on  the  cellulose  membrane,  but  still  retains  its  power 
if  setting  up  plasmolysis.    This  reaction  to  temperature  confirms  the 
iew  that  the  juice  of  the  Fungus  contains  a  soluble  ferment.    The 
suits  of  de  Bary's  researches  have  been  confirmed  and  in  part 
Lipplemented  by  the  experiments  of  Laurent1. 

It  is  a  fact  of  common  observation  that  the  Sderotinia  libertiana 
vades  for  the  most  part  young  plants.  It  may  therefore  be  asserted 
hat  the  disease  produced  by  this  Fungus  is,  like  scarlatina  or  measles 
the  human  subject,  an  "infantile"  disease.  De  Bary  suggested 
hat  the  immunity  of  adult  plants  must  depend  on  the  greater 
sistance  which  their  cell-membranes  offer  to  the  fluid  secreted  by 
|he  mycelial  filaments.  Direct  experiments  have  shown  the  accuracy 
f  his  suggestion.  Whilst  the  fluid  extracted  from  the  Sderotinia 

ily  digests  the  tissue  of  young  plants  it  leaves  intact  that  of  adult  [36] 
ilants  of  the  same  species. 

In  the  course  of  this  disease  we  have  a  struggle  going  on  between 
plants.  The  parasite  brings  into  play  toxic  and  digestive  secre- 
ions  with  which  it  seeks  to  impregnate  its  host.  The  attacked  plant 
efends  itself  by  the  secretion  of  membranes  capable  of  resisting  the 
:tion  of  the  secretions  of  the  Fungus.  This  struggle  by  means  of 
liemical  substances  is,  however,  directed  by  the  activity  of  the  living 

1  Ann.  de  VInst.  Pasteur,  Paris,  1899,  t.  xm,  p.  44. 
B.  Q 


34  Chapter  II 

cells  of  the  two  belligerent  plants,  an  activity  dependent  upon  the 
irritability  of  their  protoplasm. 

The  example  we  have  just'  studied  may  serve  as  a  type  for  our 
examination  of  the  phenomena  of  immunity  in  the  vegetable  kingdom. 
The  crux  is  above  all  to  prevent  the  access  of  the  parasites  to  the 
vital  parts  of  the  plant  by  opposing  to  them  membranes  as  resistant 
as  possible.  Consequently  the  majority  of  plants,  directly  the  smallest 
lesion  is  produced,  react  by  an  abundant  cell-proliferation  and  by  the 
suberisation  of  the  outer  layers.  The  cell-membranes  of  the  latter 
thicken,  the  cellulose  is  transformed  into  suberin  ;  a  layer  of  cork 
not  very  permeable  to  fluids  and  gases  being  thus  formed.  By 
suberisation  the  plant  reacts  against  grosser  lesions,  incisions  or 
burns,  as  well  as  against  the  decay  set  up  by  micro-organisms. 

Massart1,  in  an  extremely  interesting  memoir,  has  brought 
together  the  known  data  concerning  cicatrisation  in  plants  and  has 
demonstrated  the  fact  that  it  is  a  very  variable  process.  In  many 
leaves  after  being  damaged  there  is  no  attempt  to  react  by  forming 
cicatricial  tissue.  Many  aquatic  and  marsh  plants  react  but  feebly. 
Their  tissues  die  and  turn  brown,  the  plants  failing  to  defend  them- 
selves by  cicatrices,  probably  owing  to  the  ease  with  which  the  lost 
parts  can  be  replaced.  When,  however,  in  the  same  plants,  there  is 
produced  a  lesion  of  parts  which  are  of  great  importance  for  the 
preservation  of  the  integrity  of  the  individual  or  a  lesion  of  the  organs 
which  enable  the  plant  to  continue  its  existence  through  the  winter, 
cicatrisation  of  the  wounds  takes  place  rapidly. 

The  old  or  adult  parts  in  most  cases  react  differently  from  the 

young  parts.    Thus,  the  young  leaves  of  Clisia  (the  example  selected 

by  Massart)  react  to  traumatism  very  promptly  and  form  a  genuine 

[37]  callus  which  makes  good  the  injury,  but  the  adult  leaves  merely  pro-j 

duce  a  layer  of  cork  in  the  immediate  neighbourhood  of  the  lesion. 

The  essential  mechanism  of  cicatrisation  has  not  yet  been  satisfac-j 
torily  analysed,  but  it  is  evident,  when  all  is  said  and  done,  that  it  is 
directed  by  the  irritability  of  the  living  protoplasm  of  the  vegetable 
cells. 

Many  plants  protect  their  wounds  with  a  kind  of  dressing,  using  | 
for  that  purpose  juices  which  harden  on  exposure  to  the  air.  Some-| 
times  these  juices,  e.g.  latex,  are  preformed  in  the  plant  and  are  as  it 
were  always  ready  for  use ;  at  other  times  they  may  be  formed  only 

1  "  La  cicatrisation  chez  les  vegetaux,"  Mem.  couron.  de  I'Acad.  roy.  de  BelgiqueA 
Bruxelles,  1898,  t.  LVII. 


Immunity  in  Multicellular  Plants 


35 


as  the  result  of  an  injury.  In  this  latter  case  the  resins  and  gums 
which  serve  to  close  the  wound  and  to  protect  the  living  parts  receive 
the  name  of  "  cicatricial  secretions  "  (Wundsecrete).  According  to  the 
view  first  formulated  by  de  Vries,  those  juices  which  harden  under 
the  action  of  air  prove  of  great  service  both  as  natural  "  dressings  " 
and  as  safeguards  against  the  attacks  of  plants  and  animals.  Indeed 
many  of  these  secretions  contain  essences  whose  antiseptic  and  toxic 
action  is  now  generally  recognised1. 

The  suberisation,  the  formation  of  a  callus,  and  the  secretion  of 
juices  which  close  the  wounds,  are  all  means  readily  utilised  and  very 
potent  in  ensuring  the  resistance  of  plants  against  all  sorts  of  injurious 
influences  which  may  be  set  up  by  a  morbid  condition.  But  these 
processes  are  not  the  only  means  which  plants  have  at  their  disposal. 
The  living  elements  of  plants  usually  secrete  a  cell-juice  of  acid  re- 
action which  plays  a  very  important  part  in  the  defence  of  plants 
against  pathogenic  agents.  Laurent2  has  studied  this  phase  of  the 
immunity  of  plants  against  bacterial  decay.  A  variety  of  the  Bacillus 
coli  communis,  according  to  this  observer,  attacks  the  potato  by 
means  of  its  secretions  in  a  fashion  analogous  to  that  already  de- 
scribed when  discussing  Sclerotinia.  This  bacillus  produces  a  soluble 
ferment  which  has  the  power  of  digesting  the  cellulose  membrane  in 
the  tuber  of  the  potato,  and  at  the  same  time  secretes  an  alkaline 
|  juice  without  which  this  digestion  cannot  go  on.  Heating  to  62°  C. 
destroys  the  soluble  ferment  and  the  fluid  thus  heated  is  no  longer 
able  to  digest  the  layers  of  the  cell-membrane  between  the  cells.  In 
j  spite  of  exposure  to  this  temperature,  however,  it  still  retains  intact 
|  one  or  even  several  substances  which  may  continue  to  cause  con-  [38] 
traction  of  the  protoplasm  and  ultimately  kill  it. 

When  Laurent  placed  cut  halves  of  tubers  coming  from  races  of 
(potato  which  were  most  resistant  to  bacterial  decay  in  the  fluid  pro- 
duced by  the  Bacillus  coli  and  afterwards  inoculated  them  with  the 
bacillus  itself,  he  invariably  found  that  the  vegetable  cells  were  pro- 
foundly affected. 

The  alkaline  secretions  of  the  bacillus  studied  by  Laurent  may  be 
[neutralised  by  the  acid  juice  of  the  potato,  and  when  certain  races  of 
tubers  prove  immune  from  decay,  it  is,  according  to  this  observer, 
(because  of  the  production  of  sufficiently  acid  cell-juices.  Moreover  he 

1  Cf.  Frank,  "Die  Krankheiten  der  Pflanzen,"  Breslau,  2te  Aufl.,  1895,  Bd.  i,  S.  43. 
"Recherches  experimentales  sur  les  maladies  des  plantes,"  Ann.   de  FInst. 
\Pasteur,  Paris,  1899,  t.  xm,  p.  1. 

3—2 


36  Chapter  II 

actually  succeeded  in  communicating  an  artificial  immunity  to  varieties 
of  the  potato  which  were  most  susceptible  to  decay  by  immersing 
them  for  several  hours  in  solutions  of  certain  organic  acids.  On  the 
other  hand,  when  he  treated  varieties  endowed  with  a  well-marked 
natural  immunity  with  alkaline  solutions,  the  tubers  became  very 
susceptible  to  the  decay  set  up  by  the  bacillus. 

The  struggle  between  the  potato  and  the  Bacillus  coll  reduces 
itself,  then,  to  the  chemical  reaction  between  the  alkaline  cell-secre- 
tions of  the  micro-organism  and  the  acid  secretions  of  the  potato. 
This  general  fact,  according,  to  Laurent,  explains  the  part  played  by 
certain  manures  in  determining  the  susceptibility  or  the  resistance 
manifested  by  the  potato  and  many  other  plants  against  infective 
diseases. 

We  know  that  the  addition  of  phosphates  to  the  soil  increases  the 
immunity  of  certain  cultivated  plants.  These  substances  are  greedily 
absorbed  by  the  roots  and  produce  acid  salts  which  are  dissolved  in 
the  cell-juice.  The  nitrogenous  manures,  on  the  other  hand,  both 
potassic  and  lime,  diminish  the  resistance  of  the  same  plants, 
probably  from  the  fact  that  they  bring  about  a  diminution  of  the 
acidity  of  the  cell-juice. 

But  these  manures  can  act  differently  on  different  plants.  Thus 
the  same  phosphates  which  confer  immunity  on  the  potato  against 
bacterial  decay  render  the  Jerusalem  artichoke  more  susceptible  to 
the  attacks  of  the  Sclerotinia. 

Laurent  explains  this  fact  as  due  to  the  difference  in  the  reaction 
of  the  medium,  which  favours  the  action  of  one  or  the  other  of  the 
[39]  soluble  ferments  of  the  two  parasites.  The  ferment  of  the  bacillus 
digests  the  cell-membrane  in  an  alkaline  or  feebly  acid  medium, 
whereas  the  hyperacidity  which  results  from  the  absorption  of  the 
phosphates  prevents  this  digestion  and  consequently  aids  the  plant  in 
its  struggle.  On  the  other  hand,  the  ferment  of  Sclerotinia,  as  is  seen 
from  the  researches  of  de  Bary,  will  digest  cellulose  even  in  a  dis- 
tinctly acid  medium.  The  hyperacidity,  induced  by  the  phosphated 
manure,  in  this  case  favours  the  parasite  and  enables  it  to  gain  the 
upper  hand  in  the  struggle  with  the  tissues  of  the  artichoke. 

Iii  addition  to  neutralising  the  microbial  products  the  acids  of 
the  cell-juice  also  act  injuriously  on  most  bacteria,  which  will  only 
develop  in  neutral  or  alkaline  media;  it  is  for  this  reason  that 
bacterial  diseases  are  so  much  rarer  in  plants  than  in  animals. 

The  secretion  of   cell-juices  is  consequently  a  very  important 


Immunity  in  Multicellular  Plants  37 

clement  in  the  defence  of  plants  ;  it  will  be  useful,  therefore,  to 
ascertain  as  definitely  as  possible  the  essential  mode  of  its  action. 
Vegetable  cells  are  as  a  rule  very  sensitive  to  the  influences  to  which 
they  are  exposed;  they  distinguish  with  great  precision  the  changes 
which  take  place  in  their  surroundings.  They  are,  indeed,  capable  of 
discerning  not  only  the  physical  properties  but  also  the  chemical  com- 
position of  the  medium  in  which  they  live. 

Vegetable  cells  estimate  very  accurately  the  osmotic  pressure  of  the 
fluid  which  bathes  them,  and  they  react  towards  this  solution  by  increas- 
ing or  diminishing  their  own  internal  pressure.  Van  Rysselberghe1, 
in  an  investigation  very  carefully  carried  out,  demonstrated  that 
when  vegetable  cells  (especially  the  epidermic  cells  of  certain  species 
of  Tradcscantia)  are  placed  in  a  solution  of  greater  density  than  that 
to  which  the  cells  are  accustomed,  the  intracellular  pressure  increases ; 
in  a  solution  of  less  density  the  pressure  diminishes.  These  changes 
in  osmotic  pressure  are  due  to  variations  in  density  of  the  cell-juice, 
whilst  these  variations  are  in  turn  determined  by  chemical  transforma- 
tions. Thus,  if  the  cell  be  treated  with  a  too  concentrated  solution  it 
produces  oxalic  acid,  which  dissolving  in  the  cell-juice,  is,  owing  to  the 
smallness  of  its  molecule,  very  osmotic. 

With  the  purpose  of  confirming  this  by  exact  facts  van  Rysselberghe 
has  studied  the  acids  of  the  cell-juice  of  Tradescantia.  In  the  normal  [40] 
juice  he  found  that  malic  acid  was  constantly  present  and,  in  rare  cases 
only,  traces  of  oxalic  acid.  He  then  determined  the  acids  present  in 
the  leaves  of  the  same  plant  after  they  had  been  several  days  in  con- 
tact with  fairly  concentrated  solutions  of  cane  sugar.  In  each  analysis 
he  found  oxalic  acid  in  quite  appreciable  quantity.  There  is  then,  in 
the  plant  which  adapts  itself  to  more  concentrated  solutions  of  the 
medium,  a  production  of  oxalic  acid  which  serves  the  purpose  of 
increasing  the  pressure  of  the  cell-juice. 

The  origin  of  this  oxalic  acid  could  not  be  accurately  demonstrated, 
but  van  Rysselberghe  considers  that  it  is  probably  formed  at  the 
expense  of  the  glucose. 

According  to  the  researches  of  Giessler  oxalic  acid  is  localised 
specially  in  the  epidermis  and  generally  in  the  peripheral  tissues  of 
plants ;  it  is  very  probable,  therefore,  that  it  fulfils  a  protective  rdle 
against  all  kinds  of  injurious  influences.  Botanists  hold  indeed  that 
oxalic  acid  keeps  herbivorous  animals,  especially  slugs  and  plant  lice, 

"  Reaction  osmotique  des  cellules  vegetales,"  Mem.  couron.  de  TAcad.  roy.  de 
;    Bdgique,  Bruxelles,  1899. 


38  Chapter  II 

from  attacking  plants  that  are  rich  in  this  substance.  It  is  of  use, 
also,  in  retaining  the  moisture  in  the  superficial  cells.  It  is  very 
probable  that  it  also  plays  an  important  part  as  a  factor  in  the  main- 
tenance in  plants  of  immunity  against  bacterial  diseases. 

The  vegetable  protoplasm,  which  is  capable  of  increasing  the 
production  of  acids  in  order  to  raise  the  osmotic  pressure,  can  also,  in 
case  of  need,  cause  a  diminution. 

When  the  cells  of  Tradescantia  are  transferred  from  a  con- 
centrated solution  into  one  much  more  dilute  there  may  often  be 
observed  a  precipitation,  in  the  cell-juice,  of  crystals  of  oxalate  of 
lime ;  this  brings  about  a  diminution  in  the  osmotic  pressure.  When 
the  density  of  the  medium  is  altered,  and  the  vegetable  tissue  is  again 
transferred  to  a  stronger  solution,  the  oxalate  crystals  are  seen  to 
dissolve,  as  a  result  of  a  new  production  of  acid. 

These  chemical  processes,  so  important  to  the  life  of  plants  in 
general  and  for  ensuring  to  them  immunity  against  infective  agents 
in  particular,  are  dependent  upon  the  irritability  of  the  protoplasm. 
Imprisoned  in  its  resistant  and  more  or  less  thick  membrane,  the 
living  part  of  the  vegetable  cell  estimates  with  nice  discrimination 
every  change  that  takes  place  around  it. 

[41]  Massart1  has  shown  that  the  stimulation  produced  by  traumatism 
is  often  propagated  a  considerable  distance  and  may  excite  a  reaction 
in  very  remote  cells.  If  the  mid-rib  of  a  leaf  of  Impatiem  sultani 
be  cut  near  the  base  of  the  limb  the  wound  does  not  cicatrise  but,  a 
few  days  later,  the  leaf  becomes  detached  from  the  stem. 

Irritability  is  a  fundamental  property  of  all  living  beings.  The 
plant  may  react  by  rapid  movements,  as  in  the  case  of  the  Mimosa 
pudica,  or  more  slowly — by  chemical  reactions — as  in  the  case  of 
adaptation  to  density  of  medium.  These  reactions  are  produced  as 
the  result  of  various  irritabilities  which  exhibit  a  specific  character. 

It  is  this  specificity  that  determines  whether  the  reaction  that  is 
manifested  by  the  movements  shall  be  produced  in  this  direction  or 
in  that.  The  stem,  owing  to  the  specific  irritability  of  its  living  parts, 
turns  to  the  light ;  whilst  the  root,  guided  by  a  different  irritability, 
grows  down  into  the  soil. 

The  irritability  of  plants,  like  that  of  unicellular  organisms,  is 
subject  to  the  psycho-physical  law  of  Weber-Fechner.  Pfeffer2  first 


1  "La  cicatrisation,"  I.e.,  p.  61. 

2  Untersuch.  a.  d.  lotan.  Inst.  zu  Tubingen,  Leipzig,  1884,  Bd.  i,  S.  363. 


Immunity  in  Multicellular  Plants  39 

demonstrated  this  for  the  motile  spermatozoids  of  the  Cryptogams. 
Massart1,  by  a  series  of  ingenious  experiments  on  the  irritability 
of  a  Mould  (Pliycomyces  nitens)  to  light,  has  shown  that  the  same  law 
regulates  the  movements  of  this  plant  towards  the  source  of  light. 
This  irritability  of  the  Fungus  to  light  is  much  more  delicate  than  is 
the  chemiotaxis  of  the  spermatozoids  of  the  Mosses  and  the  Ferns 
and  than  that  of  the  Bacteria. 

Errera  concluded  from  a  consideration  of  the  experiments  of 
van  Rysselberghe  that  the  osmotic  reaction  of  plants  must  also  come 
under  this  psycho-physical  law.  His  pupil  at  his  request  made 
systematic  researches  on  the  subject  and  the  results  have  entirely 
confirmed  his  prevision.  According  to  the  data  obtained  by  van 
Rysselberghe2,  the  cellular  osmotic  reaction  increases  in  arithmetical 
progression  as  the  osmotic  stimulation  increases  in  geometrical  pro- 
gression. The  osmotic  reaction  is  therefore  proportional  to  the 
logarithm  of  the  stimulation. 

To  sum  up,  the-  phenomena  of  adaptation  and  of  immunity  in  plants  [42] 
are,  as  in  the  unicellular  organisms,  very  widely  distributed.  Plants 
defend  themselves  by  means  of  their  resistant  membranes  and  by 
secretions  whose  physical  and  chemical  properties  they  are  able  to 
modify.  These  phenomena  are  dependent  on  the  living  parts  of  the 
cell  which  regulate  them  according  to  their  greatly  developed 
irritabilities.  Thanks  to  this  power,  plants  can  gradually  adapt  them- 
selves to  concentration  of  the  medium  and  to  the  presence  of  poisons 
which,  at  first,  set  up  serious  disturbances.  Plants  therefore,  along- 
side a  natural  immunity,  possess  an  acquired  immunity  against  many 
pathogenic  agents. 

1  "  Recherches  sur  les  organismes  inferieurs,"  Bull,  de  VAcad.  de  Belgique,  1888, 
2*  serie,  t.  xvi,  v,  12. 

2  Z.c.,  p.  40. 


[43]  CHAPTER    III 

PRELIMINARY  REMARKS  ON  IMMUNITY  IN  THE 
ANIMAL  KINGDOM 

Examples  of  natural  immunity  among  the  Invertebrates. — Immunity  against  micro- 
organisms and  insusceptibility  to  microbial  poisons  are  two  distinct  properties. — 
The  refractory  organism  does  not  eliminate  micro-organisms  by  the  excretory 
channels. — It  destroys  them  by  a  process  of  resorption. — The  fate  of  foreign 
bodies  in  the  organism. — The  resorption  of  cells. — Intracellular  digestion. — 
This  digestion  effected  by  the  aid  of  soluble  ferments. — Digestion  in  Planarians 
and  Actinians. — Actino-diastase. — Transition  from  intracellular  digestion  to 
digestion  by  secreted  juices. — Digestion  in  the  higher  animals. — Enterokynase 
and  the  part  it  plays  in  digestion. — The  psychical  and  nervous  elements 
in  digestion. — Adaptation  of  the  pancreatic  secretion  to  the  kind  of  food. — 
Excretion  of  pepsin  in  the  blood  and  in  the  urine. 

As  shown  in  the  two  preceding  chapters  unicellular  organisms 
and  plants  afford  evidence  of  numerous  phenomena  of  immunity. 
Alongside  natural  immunity  we  find  in  them  undoubted  evidence  of 
an  adaptation  to  the  presence  of  morbific  agents,  evidence  which 
warrants  us  in  inferring  that  cases  of  acquired  immunity  are  frequent. 
This  being  the  case  it  is  quite  natural  that  the  animal  kingdom  should 
be  no  exception  to  the  general  rule.  Indeed,  immunity  against  patho- 
genic agents  is  widely  distributed  in  animals,  and  we  continually  see 
manifestations  of  natural  immunity  not  only  against  parasites  and 
their  toxins,  but  against  poisons  in  general.  Just  as  frequently  we 
find  cases  of  acquired  immunity  against  these  morbific  agents. 

As  yet  we  know  but  little  concerning  the  phenomena  of  immunity 
in  the  lower  animals  belonging  to  the  great  group  of  the  Invertebrata. 
But  it  may  be  affirmed  with  certainty  that  these  also  are  often 
endowed  with  a  natural  immunity  against  micro-organisms  and 
bacterial  toxins.  As  an  example  I  may  cite  the  case  of  the  large 
white  larvae  of  the  Rhinoceros  beetle  (Oryctes  nasicornis)  frequently 
met  with  in  tanner's  bark.  Very  susceptible  to  the  cholera  vibrio- 
s' of  a  culture1  of  this  organism  being  sufficient  to  set  up  a  fatal 

1  [Probably  a  surface  growth  on  a  sloped  agar  tube  (Transl.).] 


Preliminary  remarks  on  immunity  in  animal  kingdom  41 

septicaemia  —  these  larvae  exhibit  a  very  remarkable  natural  immunity  [44] 
against  the  bacilli  of  anthrax  and  diphtheria.  A  large  dose  of  bacteria 
of  the  second  anthrax  vaccine,  fatal  to  rabbits,  guinea-pigs  and  mice, 
is  borne  without  any  inconvenience  by  the  larvae  of  the  Rhinoceros 
beetle.  They  are  equally  refractory  to  large  doses  of  the  diphtheria 
bacillus.  And  yet,  there  are  not  wanting  species  of  insects  which 
are  susceptible  to  these  same  micro-organisms.  Thus,  according 
to  A.  Kovalevsky1,  crickets  contract  anthrax  very  readily  even  at 
moderate  temperatures  (22°  —  23°  C.).  On  the  other  hand  they  are, 
according  to  the  same  author,  refractory  to  the  bacillus  of  avian 
tuberculosis.  Many  of  the  Invertebrata,  studied  from  this  point  of 
view,  present  analogous  facts,  with  which,  however,  we  need  not  at 
present  occupy  ourselves. 

In  the  Vertebrata  in  general  and  in  Man  in  particular,  natural 
immunity  against  many  infective  diseases  and  soluble  poisons  is 
so  widespread  that  we  are  at  no  loss  to  find  examples  for  citation. 
We  have  a  whole  series  of  human  infections  whose  study  is  rendered 
particularly  difficult  simply  because  of  the  natural  immunity  of  all 
other  species  of  animals  from  these  infections.  Such  are  syphilis, 
scarlatina,  leprosy,  exanthematous  typhus,  etc.  On  the  other  hand, 
a  large  number  of  diseases,  very  infective  for  domestic  animals,  are 
quite  innocuous  to  man.  In  this  group  we  have  cattle  plague, 
strangles,  contagious  pleuro-pneumonia,  fowl  cholera,  pneumo- 
enteritis  of  pigs,  and  a  number  of  other  diseases. 

As  in  a  very  large  majority  of  instances  pathogenic  organisms  act 
through  the  agency  of  their  toxic  products,  one  might  believe  —  and 
this  has  been  assumed  repeatedly  —  that  natural  immunity  against 
infective  diseases  is  dependent  on  the  insusceptibility  of  the  refractory 
organism  to  the  specific  poisons. 

Such  a  supposition  cannot  survive  criticism.  We  have  un- 
doubted instances  of  a  species  of  animal  being  resistant  both  to 
a  micro-organism  and  to  its  toxin.  Such  instances,  however,  are 
rare  and  usually  an  organism  that  is  refractory  or  only  slightly 
susceptible  to  the  micro-organism  itself  is  very  susceptible  to  its 
toxic  products.  Even  those  micro-organisms  which  come  almost 
constantly  in  contact  with  the  human  organism  without  becoming 
pathogenic,  may  produce  toxins  capable  of  gravely  affecting  health. 


" 


Etude  experimental  sur  les  glandes  lymphatiques  des  invertebres,"  Melanges 
biol.  de  FAcad.  d.  sc.  de  St-  Peter  sb.,  1894,  t,  xin,  p.  458. 


42  Chapter  III 

[45]  Let  us  take  as  an  example  the  bacillus  of  blue  pus.  This  organism  is 
most  widely  diffused  in  human  surroundings.  According  to  Schimmel- 
busch1  it  is  met  with  on  the  skin  of  the  arm-pits  and  of  the  inguinal 
region  of  one-half  of  mankind.  From  the  skin  it  very  often  passes 
into  the  dressings  of  wounds  which  then  assume  the  characteristic 
and  so  long  recognised  blue  colour.  The  same  bacillus  is  also  found 
in  the  intestines  of  both  sick  and  healthy  persons.  Jakowski2  has 
met  with  it  in  the  faeces  coming  from  intestinal  fistulae  in  two  women 
who  had  undergone  operations.  Now,  in  spite  of  these  specially 
favourable  conditions  for  the  production  of  infection,  the  Bacillus 
pyocyaneus  has  remained  harmless.  It  is  only  in  children,  and  even 
then  rarely,  that  it  can  be  convicted  of  exciting  disease.  Man,  then, 
usually  enjoys  a  true  natural  immunity  against  the  Bacillus  pyo- 
cyaneus. And  yet  it  is  not  to  his  insusceptibility  to  the  pyocyanic 
toxin  that  he  is  indebted  for  this  immunity.  Schaffer3,  having  in- 
jected himself  in  the  shoulder  witli  half  a  c.c.  of  a  sterilised  culture 
of  B.  pyocyaneus,  developed  fever  and  an  erysipelatous  swelling. 
Bouchard  and  Charrin4  injected  pyocyanic  toxin  into  patients  who 
reacted  with  more  or  less  fever  and  by  other  toxic  symptoms. 

Another  extremely  common  saprophyte,  the  Micrococcus  pro- 
digiosus,  is  incapable  of  setting  up  an  infective  disease,  but  this 
does  not  prevent  its  products  from  exercising  a  toxic  action,  often 
very  grave,  in  man.  The  frog,  which  is  refractory  to  the  cholera 
vibrio,  undergoes  a  fatal  intoxication  when  cholera  toxin  is  injected. 
One  of  the  most  striking  examples  is  furnished  in  the  case  of  the 
human  tubercle  bacillus  and  tuberculin.  Man  is  much  more  resistant 
than  is  the  guinea-pig  to  the  pathogenic  action  of  this  organism, 
yet  he  is  incomparably  more  susceptible  to  its  toxin  (tuberculin). 
According  to  the  researches  of  Behririg  and  Kitashima5,  the  sheep,  of 
all  species  of  mammals,  is  most  susceptible  to  the  tubercular  poison ; 
the  Bovidae  and  the  guinea-pig  occupy  an  inferior  rank  in  the  scale 
of  susceptibility.  On  the  other  hand,  the  guinea-pig  is  very  sus- 
ceptible to  the  tubercle  bacillus ;  the  Bovidae  are  less  so  and  the 

[46]  sheep  is  still  more  resistant  to  tuberculosis.    It  is  unnecessary  to 
multiply  instances.    Immunity  against  microbial  infection  and  against 

1  "Ueber  griinen  Eiter,"  Volkmann's  Samml.  klin.  Vortr.,  No.  62,  Leipzig,  1893. 

2  "Processus  chimiques  dans  les  intestins  de  1'homme,"  Arch.  d.  sc.  biol.    de 
St-Petersb.,  1892,  t.  I,  p.  539;  Ztschr.  f.  Hyg.,  Leipzig,  1893,  Bd.  xv,  S.  474. 

3  Cited  by  Schimmelbusch,  I.e. 

4  Compt.  rend.  Acad.  d.  Sc.,  Paris,  1892,  t.  II,  p.  1226. 
6  Berl.  klin.  Wchnschr.,  1901,  S.  163. 


Preliminary  remarks  on  immunity  in  animal  kingdom  43 

intoxication  are  two  distinct  properties,  so  that  it  is  impossible  to 
reduce  the  former  to  an  insusceptibility  to  toxins.  We  must  there- 
fore consider  these  two  kinds  of  immunity  separately  and  we  will  first 
consider  the  resistance  of  the  animal  organism  against  living  infective 
micro-organisms. 

Refractory  human  beings  and  animals  may  be  inoculated  with 
a  large  number  of  micro-organisms  without  being  affected.  Thus 
Opitz1  injected  10,000,000  organisms  into  the  blood  of  a  dog. 
Twenty  minutes  later  he  could  find  no  more  than  9000.  It  is  then 
quite  natural  to  ask,  What  becomes  of  these  micro-organisms  after 
they  have  made  their  way  into  the  interior  of  the  refractory  organism? 
It  has  been  suggested  that  the  animal  gets  rid  of  the  pathogenic 
germs  much  as  it  does  of  all  kinds  of  soluble  poisons.  Certain  of 
these  poisons,  such  as  iodine  and  alcohol,  are  in  great  part  eliminated 
by  the  kidneys  ;  others,  such  as  iron,  by  the  alimentary  canal.  Why, 
it  is  asked,  should  not  micro-organisms  also  be  eliminated  by  the 
same  channels?  Fliigge  has  adopted  this  view  and  has  expounded 
it  in  his  work  on  ferments  and  micro-organisms2.  Moreover  he 
suggested  to  Wyssokowitch3  that  he  should  carry  out  a  large  series 
of  experiments  with  the  object  of  verifying  this  theory.  But  numerous 
very  careful  researches  have  given  a  result  quite  at  variance  with  the 
forecast  made  by  Fliigge.  Micro-organisms  of  various  species,  injected 
into  the  blood-vessels  of  rabbits  and  dogs,  were,  in  those  cases  where 
these  animals  are  refractory,  never  eliminated,  either  by  the  kidneys 
or  by  any  other  of  the  excretory  channels  which  were  studied.  When 
bacteria  pass  into  the  secretions,  lesions  of  the  tissues,  more  or  less 
grave,  are  invariably  present. 

This  result  has  been  repeatedly  confirmed  and  has  been  accepted 
as  a  general  experience.  The  elimination  of  micro-organisms  by  the 
urine  indicates  not  merely  the  absence  of  immunity,  but  implies, 

1  also,  a  susceptibility  of  the  organism.    In  many  septicaemias,  such 
as  those  produced  by  the  anthrax  bacillus,  the  streptococcus  and 
other  bacteria,  or  in  less  generalised  diseases,  such  as  typhoid  fever, 
bacteria  are  found  in  the  urine,  often  in  large  numbers.    In  these  [47] 
cases  it  is  a  question  of  anything  but  a  refractory  condition  even  of 

i  the  slightest  degree. 

1  Ztschr.f.  Hyg.,  Leipzig,  1898,  Bd.  xxix,  S.  548. 

"  Fermente  und  Mikroparasiteu  "  in  Ziemssen  u.  Pettenkofer's  "  Handbuch  der 
Hygiene,"  Leipzig,  1883. 

3  "Ueber  die  Schicksalc  der  in's  Blut  injicirten  Mikroorganismen,"  Ztschr.  f. 
Hyg.,  Leipzig,  1886,  Bd.  I,  S.  1. 


44  Chapter  III 

In  recent  years,  however,  several  works  have  been  published 
the  aim  of  which  was  to  demonstrate  the  inaccuracy  of  this 
apparently  well-established  thesis.  Biedl  and  Kraus1  in  Vienna 
took  the  initiative  and  announced  in  a  detailed  work  that  micro- 
organisms can  readily  pass  intact  into  the  kidney  and  that  this  organ 
in  virtue  of  its  physiological  function  eliminates  them.  The  organisms 
were  said  to  leave  the  blood  capillaries  by  the  normal  process  of 
diapedesis  and  were  then  eliminated  with  the  urine.  The  liver  in 
a  physiological  condition,  according  to  the  researches  of  these 
authors,  is  equally  capable  of  allowing  of  the  passage  of  micro- 
organisms ;  indeed  it  aids  in  discharging  them  from  the  system.  On 
the  other  hand,  the  pancreas  and  the  salivary  glands  were  incapable 
of  fulfilling  this  function.  Von  Klecki2  obtained  similar  results. 
He  also  holds  that  the  kidney  is  the  principal  organ  of  elimination 
for  micro-organisms  which  have  penetrated  into  a  refractory  organism. 

With  these  contradictions  before  him,  Opitz3  set  himself  to  study 
this  question  in  Fliigge's  laboratory  at  Breslau.  Having  critically 
reviewed  the  technical  methods  of  his  predecessors  and  carried  out 
a  series  of  new  experiments,  he  declared  categorically  "that  a 
physiological  excretion,  by  the  kidneys,  of  the  micro-organisms  which 
circulate  in  the  blood,  does  not  exist."  For  Opitz  "the  frequent 
appearance  of  micro-organisms  in  the  urine  of  animals  into  whose 
blood,  a  short  time  previously,  living  bacteria  have  been  injected,  is 
due  to  mechanical  and  chemical  lesions  of  the  vessel  wall  and  of  the 
renal  epithelia." 

This  question  might  be  looked  upon  as  definitely  settled  in 
favour  of  the  first  results  obtained  by  Wyssokowitch  were  it  not 
that  other  voices  had  been  raised  in  favour  of  a  physiological 
excretion  of  the  micro-organisms  by  the  renal  channels.  Pawlowsky4 
has  recently  published  a  'long  work  on  this  subject  in  which  he 
attempts  to  demonstrate  that  certain  micro-organisms^  even  when 
introduced  into  the  subcutaneous  tissue  of  animals,  pass  very  rapidly 
[48]  (at  the  end  of  a  quarter  of  an  hour)  into  the  uropoietic  organs  and 
are  eliminated  with  the  urine. 

It  was  necessary  to  put  an  end  to  these  controversies  and  Me  tin5 
undertook  a  series  of  researches  at  the  Pasteur  Institute  with  the 

1  Ztschr.f.  Hyg.,  Leipzig,  1897,  Bd.  xxvi,  S.  353. 

2  Arch./,  exper.  Path.,  Leipzig,  1897,  Bd.  xxxix,  S.  39. 

3  Ztschr.f.  Hyg.,  Leipzig,  1898,  Bd.  xxix,  S.  52$. 

4  Ztschr.f.  Hyg.,  Leipzig,  1900,  Bd.  xxxin,  S.  261. 

6  Ann.  de  VInst.  Pasteur,  Paris,  1900,  t.  Xiv,  p.  415. 


Preliminary  remarks  on  immunity  in  animal  kingdom  45 

object  of  clearing  up  this  question.  He  guarded  himself  against  the 
objections  justly  made  against  his  predecessors  and  conducted  his 
experiments  under  unexceptionable  conditions.  He  injected  several 
species  of  micro-organisms  into  the  veins  of  rabbits  and  into  the 
subcutaneous  tissue  of  guinea-pigs.  At  various  intervals  he  per- 
formed laparotomy  on  these  animals,  pulled  out  the  bladder  and 
drew  off  the  urine  in  such  a  fashion  that  no  trace  of  blood  could 
get  into  it.  The  results  were  most  conclusive.  Xever,  when  the 
experiment  was  conducted  under  the  rigorous  conditions  just  men- 
tioned, did  the  micro-organisms  traverse  the  kidneys  of  resistant 
animals  nor  were  they  ever  met  with  in  their  urine. 

Metin's  researches  on  the  passage  of  micro-organisms  through  the 
liver  in  refractory  animals  gave  the  same  results.  In  no  case  was  he 
able  to  find  in  the  bile  any  of  the  organisms  that  had  been  injected 
into  the  blood  or  under  the  skin.  At  the  end  of  his  memoir 
Met  in  sums  up  his  results  as  follows :  "  (1)  The  kidneys  and  the 
liver  are  impermeable  to  bacteria  introduced  into  the  organism, 
subcutaneously  or  intravenously  ;  (2)  when  the  culture  tubes  contain 
colonies  of  the  injected  micro-organism,  it  is  because  there  has  been 
a  certain  amount  of  blood  in  the  fluid  inoculated,  this  being  an 
indication  of  a  vascular  or  epithelial  lesion,  either  mechanical  or 
chemical."  We  were  present  at  M.  Metin's  experiments  and  can  bear 
witness  to  their  exactitude. 

There   can  no  longer  be  any  doubt  then  on  this  point.    The 
elimination    of   the   micro-organisms    from    the    refractory    animal 
takes  place,  as  indicated  in  Wyssokowitch's  first  investigation,  neither 
by  the  kidneys  nor  by  the  liver.    Some  observers  have  asserted  that 
this  elimination  may  take  place  by  the  sudoriparous  glands.    Thus, 
Brunner1  made  experiments  with  young  pigs  and  cats  into  which  he 
had   previously  injected  micro-organisms,  for  the  most  part  patho- 
j  genie.    Then  producing  a  transpiration  by  means  of  pilocarpin,  he 
[  "cultivated"  the  sweat  and  noted  the  development  of  the  same 
bacteria  as  he  had  introduced  into  the  blood.    In  a  single  experiment 
with  a  saprophyte  (Coccobacillus  prodigiosns)  he  obtained  a  positive  [49] 
result,  from  which  he  concludes  that  the  refractory  animal  gets  rid  of 
;  bacteria  which  circulate  in   its  blood  by  way  of  the  sudoriparous 
1  glands.     It  is  scarcely  allowable  to  draw  any  conclusion  from  this 
experiment  from  the  fact  that  the  snout  of  the  pig,  the  seat  of  the 
transpiration,  is  very  liable  to  small  vascular  lesions  which  might 
1  Bert.  klin.  Wchnschr.,  1891,  S.  505. 


46  Chapter  III 

furnish  the  bacteria  that  developed  on  Brunner's  plates.  Neverthe- 
less, even  in  the  case  of  pathogenic  organisms,  which  swarm  in  the 
blood,  the  sweat  .is  usually  free  from  them.  This  has  been  shown  by 
Krikliwy1  in  the  case  of  cats  inoculated  with  anthrax  whose  sweat, 
in  spite  of  the  passage  of  numerous  bacteria  into  the  circulation, 
contained  none. 

Micro-organisms,  then,  after  their  entrance  into  the  refractory 
animal,  are  not  eliminated  by  any  of  the  excretory  channels  which 
serve  for  the  elimination  of  many  of  the  soluble  poisons.  It  was 
necessary  therefore  to  seek  some  other  process  capable  of  affording 
an  explanation  of  the  disappearance  of  the  micro-organisms  which  so 
often  and  by  such  varied  means  make  their  way  into  the  interior  of 
a  resistant  organism.  For  it  is  a  well-established  fact  that  in  these 
cases  the  micro-organisms  do  disappear  completely.  This  has  been 
observed  so  often  that  it  is  unnecessary  to  offer  any  demonstration  of 
the  fact.  Perhaps  in  the  refractory  organism  the  micro-organisms 
undergo  the  fate  of  the  foreign  bodies  which  penetrate,  or  which  are 
introduced,  into  the  circulation.  It  has  long  been  known,  thanks 
especially  to  the  work  of  Hoffmann  and  Recklinghausen2,  and  of 
Ponfick3,  that  particles  of  carmine  or  vermilion  when  injected  into 
the  blood  are  deposited  in  several  organs.  They  are  found  in  the 
spleen,  the  lymphatic  glands  and  the  bone-marrow.  A  certain  number 
of  these  foreign  particles  may  even  be  fixed  in  the  liver  and  kidneys, 
but,  instead  of  passing  into  the  bile  and  the  urine,  they  remain 
lodged  in  the  interstitial  tissue  of  the  organs.  The  observers  just 
cited  noted  that  the  coloured  granules  do  not  remain  long  in  either 
the  blood  or  the  lymph  but  will  be  found  in  the  interior  of  the 
cellular  elements.  These  granules  persist  for  weeks  without  any 
appreciable  modification,  differing  in  this  from  the  micro-organisms 
which,  as  a  rule,  after  several  days  or  even  after  a  few  hours,  disappear 
from  the  refractory  organism.  This  disappearance  might  be  more 
justly  compared  to  the  resorption  of  corpuscular  elements  which 
[50]  results  in  a  more  or  less  complete  atrophy.  The  facts  concerning 
the  resorption  of  pus,  of  extravasated  blood,  of  the  mucosa  of  the 
uterus  in  pregnancy,  etc.,  have  long  been  known,  and  it  is  among 
these  that  one  should  seek  analogies  with  the  disappearance  of  the 
micro-organisms.  When  bacteria  of  various  species  are  injected  into 

1  Vratch  (in  Russian),  St  Petersburg,  1896,  NQS.  8,  12. 

2  Centralblf.  d.  med.  Wissensch.,  Berlin,  1867,  No.  31. 
8  Virchow's  Archiv,  1869,  Bd.  XLVIII,  S.  1. 


Preliminary  remarks  on  immunity  in  animal  kingdom  47 

refractory  or  not  very  susceptible  animals,  we  always  observe  a  local 
reaction  in  the  form  of  inflammation,  accompanied  by  the  appearance 
of  white  corpuscles.  Gradually  the  organisms  disappear  from  the 
point  at  which  they  are  introduced ;  the  exudation  becomes  sterile 
and  ultimately  is  completely  absorbed.  Numerous  researches,  which 
will  be  set  forth  in  the  succeeding  chapters,  have,  indeed,  demon- 
strated the  remarkable  analogy  that  exists  between  the  disappearance 
of  the  micro-organisms  from  the  refractory  animal  and  the  resorption 
of  corpuscular  elements  or  of  animal  cells. 

The  analysis  of  the  phenomena  of  this  resorption  will  help  us 
considerably  in  our  study  of  immunity  against  micro-organisms. 
When  in  any  part  of  the  animal  organism  a  collection  of  pus,  an 
effusion  of  blood,  or  any  other  organic  lesion  is  produced,  these  lesions 
are  usually  repaired  after  the  lapse  of  a  longer  or  shorter  interval. 
In  those  cases  where  the  cells  retain  their  integrity,  they  are  taken 
into  the  lymphatic  vessels  and  then  pass  into  the  circulating  blood. 
In  the  course  of  his  researches  on  the  transfusion  of  blood,  Hayem1 
observed  "that  blood  injected  into  the  peritoneum  is  absorbed  un- 
altered and  passes  with  its  anatomical  elements  into  the  general 
circulation."  He  was  able  to  demonstrate  "that  the  lymphatic 
channels  play  an  important  part  in  this  absorption."  Lesage  of 
Alfort2  confirmed  this  result.  He  found  that  in  the  dog  "  one  hour 
after  an  abundant  haemorrhage  into  the  peritoneum,  induced  ex- 
perimentally, the  red  corpuscles  commenced  to  pass  freely,  without 
alteration  and  in  very  large  numbers,  into  the  thoracic  duct."  I  have 
observed  a  similar  resorption  of  the  red  blood  corpuscles  of  the 
guinea-pig  when  injected  into  the  peritoneal  cavity  of  other  indi- 
viduals of  the  same  species.  The  white  corpuscles  can  also  be  taken 
up  by  the  lymphatic  vessels  without  being  modified  in  any  way.  At 
the  end  of  an  inflammatory  reaction  of  feeble  intensity,  set  up  in 
cold-blooded  animals,  especially  in  the  tadpole,  the  direct  passage 
of  leucocytes  from  the  exudation  into  the  lymphatic  system  may  be 
observed. 

The  examples  I  have  just  cited  are,  however,  quite  exceptional.  [51] 
In  the  great  majority  of  cases  the  cellular  elements  that  are  under- 
going resorption  are  seized  by  the  amoeboid  cells  and  are  taken  into 
their  substance.     Even  in  the  resorption  of  the  red  corpuscles,  lying 
free  in  the  peritoneal  cavity  of  the  same  species  of  animal,  a  certain 

1  Compt.  rend.  Acad.  d.  Sc..  Paris,  1884,  t.  XCVHI,  p.  749. 

2  Compt.  rend.  Soc.  de  biol,  Paris,  1900,  p.  553. 


48  Chapter  III 

Dumber  of  the  globules  do  not  pass  directly  into  the  circulation  but 
are  first  ingested  by  the  amoeboid  elements.  This  fact  is  insisted  upon 
by  Lesage.  In  inflammatory  exudations  the  leucocytes  also  become 
the  prey  of  their  fellows.  The  ingested  white  corpuscles  may  be 
recognised  for  some  time  lying  in  the  interior  of  other  leucocytes ; 
they  are  soon  broken  up,  however,  and  finally  disappear  completely. 
When,  instead  of  isolated  cells  such  as  leucocytes,  we  introduce 
fragments  of  tissues  or  of  organs  into  any  part  of  the  organism,  the 
same  mode  of  resorption  may  always  be  observed.  The  introduced 
fragments  are  first  surrounded  and  infiltrated  by  amoeboid  cells  and 
are  then  taken  up  into  their  interior. 

The  mode  of  absorption  just  described  is  very  general.  It  applies 
to  all  kinds  of  cells  and  is  observed  in  the  absolutely  normal  organism, 
as  well  as  in  a  large  number  of  pathological  conditions.  For  more 
than  fifty  years,  the  existence  of  cells  which  contain  red  blood  cor- 
puscles ("  blutkorperchenhaltige  Zellen"  of  German  writers)  has 
been  recognised ;  they  were  met  with  in  the  spleen,  the  lymphatic 
glands  and  in  many  pathological  products.  For  long  we  could  not 
explain  how  the  red  corpuscles  come  to  be  inside  other  cells. 
Virchow1  thought  that  they  got  there  as  the  result  of  a  mechanical 
pressure.  Later  histologists  succeeded  in  determining  the  true 
nature  of  cells  containing  red  blood  corpuscles  and  in  recognising 
that  the  leucocytes  had  really  ingested  the  corpuscles.  There  has 
been  much  discussion,  also,  on  the  presence  of  leucocytes  in  the 
interior  of  large  cells  in  exudations.  It  was  thought  that  these  were 
mother-cells  which  contained  a  new  generation  of  small  cells.  Writers 
even  described  a  fusion  between  the  large  cell  and  those  found  inside 
it ;  but  Bizzozero2  first  recognised  that  the  former  was  an  amoeboid 
[52]  cell  which  had  ingested  pus  corpuscles.  Since  this  observation  was 
made  numerous  cases  have  been  described  in  which  different  cell 
elements  have  been  found  in  the  large  cells.  There  could  no  longer 
be  any  hesitation  in  interpreting  these  cases  as  instances  of  ingestion 
by  leucocytes  or  similar  cells. 

The  changes  that  the  ingested  elements  undergo  within  amoeboid 
cells  may  be  compared  with  those  that  take  place  in  intracellular 
digestion.  If  the  modifications  of  the  particles  ingested  by  the  Amoebae 
be  studied  side  by  side  with  those  which  take  place  in  ingested 

1  Firchow's  Archie,  1852,  Bd.  iv,  S.  536. 

2  "Handb.  d.  klin.  Mikroskopie,"  1887,  S.  108;  Gaz.  medi  lomlarda,  1871  and 
1872  ;  Wien.  medic.  Jahrbiicher,  1872,  S.  160. 


Preliminary  remarks  on  immunity  in  animal  kingdom  49 

cells  in  the  process  of  resorption,  a  striking  analogy  may  be  observed. 
To  establish  this  satisfactorily  it  is  essential  to  begin  with  a  study 
of  intracelltilar  digestion  properly  so  called,  especially  as  in  this 
phenomenon  we  have  the  fundamental  basis  of  the  whole  of  the  theory 
developed  in  this  work. 

In  our  first  two  chapters  we  have  already  cited  examples  of  this 
intra cellular  digestion  in  the  Protozoa  (Amoebae,  Infusoria,  etc.)  and 
in  the  plasmodium  stage  of  the  Myxomycetes.  In  all  these  cases  it 
goes  on  in  the  organism,  in  a  distinctly  acid  medium,  by  the  aid  of 
ferments  which  could  be  demonstrated  in  the  Amoebae  and  Myxo- 
mycetes, and  which  are  analogous  sometimes  with  trypsin,  sometimes 
with  pepsin. 

In  the  lower  Invertebrate  we  find  the  principal  source  of  our 
knowledge  of  intracellular  digestion  in  the  digestive  organs.  This 
form  of  digestion  is  met  with  in  Sponges,  in  the  whole  of  the  Coelen- 
terates  (Medusae,  Siphonophora,  Ctenophora,  etc.),  in  the  great 
majority  of  the  Turbellaria  (Planarians,  Rhabdocoela),  and  in  certain 
of  the  Mollusca  (the  lower  Gasteropods).  In  the  Invertebrata  higher 
in  the  animal  scale,  intracellular  digestion  in  the  digestive  organs 
becomes  more  and  more  rare,  and  sometimes  it  manifests  itself  only 
in  the  larval  condition  (Phoronis) ;  ultimately  it  gives  place  perma- 
nently to  digestion  by  juices  secreted  into  the  gastro-intestinal  canal. 

In  his  sketch  of  the  comparative  physiology  of  digestion, 
Krukenberg1  sought  to  establish  two  types :  protoplasmic  or  cellular 
digestion  and  secretory  digestion.  The  former  is  effected,  according 
to  this  observer,  by  a  vital  action  independently  of  any  production  of 
soluble  ferments.  Secretory  digestion  alone,  characteristic  of  the 
Vertebrates  and  of  almost  all  the  higher  Invertebrates,  is  effected  by 
means  of  these  ferments  (diastases  or  enzymes).  Many  observers, 
adopting  this  view,  maintain  that  intracellular  digestion  presents  [53] 
a  purely  vital  phenomenon  essentially  different  from  that  of  chemical 
digestion  due  to  juices  containing  soluble  ferments  secreted  in  the 
gastro-intestinal  canal.  That  this  theory  is  absolutely  erroneous  the 
Lcceeding  pages  of  this  work  will  furnish  ample  proof. 

The  Protozoa,  from  their  small  size,  are  unsuitable  for  researches 

the  essential  phenomena  of  intracellular  digestion.  Amongst  animals 
ligher  in  the  scale  the  Planarians  lend  themselves  most  readily  to  the 
•bservation  of  this  process.  These  flat  worms  are  very  common  in 

>th  fresh  and  sea  water  and  are  easily  fed  in  captivity.    They  are 
1  "  Grundziige  einer  vergl.  Physiologie  der  Verdauung,"  Heidelberg,  1882. 
B.  4 


50 


Chapter  III 


very  voracious  animals  and,  among  other  things,  devour  the  blood  of 
man  or  animals  with  avidity.  One  has  merely  to  allow  them  to  fast 
for  a  few  days,  and  then  to  give  them  a  drop  of  blood  in  order  to  see 
their  digestive  canal  fill  itself  with  this  fluid 
(fig.  6).  The  white  Planarian,  Dendrocoelum 
lacteum,  is  well  adapted  for  these  researches. 
In  a  worm  that  has  sucked  blood  from  a 
Vertebrate,  owing  to  its  great  transparency, 
the  whole  length  of  its  intestine  with  its 
numerous  ramifications  may  be  seen.  For 
some  time  this  organ  remains  of  a  bright 
red  colour,  but  gradually  the  tinge  becomes 
brownish  or  faintly  violet.  These  changes 
of  colour  recall  those  observed  in  effusions 
of  blood  in  or  under  the  human  skin  result- 
ing from  contusions.  A  microscopical  ex- 
amination of  Planarians  that  have  been  fed 
with  blood  shows  that  the  coloration  of  their 
digestive  canal  is  due  to  red  blood  cor- 
puscles in  different  stages  of  digestion.  Im- 
mediately after  the  taking  in  of  the  blood  by 
the  Planarian  all  the  red  blood  corpuscles 
are  ingested  by  the  epithelial  cells  of  the  in- 
testine. Connected  with  the  wall  by  slender 

[54]  stalks,  these  elements  appear  as  large  amoeboid  cells  whose  free  end 
projecting  into  the  lumen  of  the  intestine  sends  out  protoplasmic  pro- 
cesses which  seize  the  red  blood  corpuscles  and  convey  them  into  the 
interior  of  the  cell.  This  goes  on  very  rapidly,  and  in  a  very  short 
time  all  the  red  corpuscles  are  found  within  the  epithelial  cells. 
As  a  result  of  the  increase  in  volume  of  these  cellular  elements 
the  intestinal  cavity  is  completely  occluded. 

Once  inside  the  cells  of  the  intestine  the  red  blood  corpuscles 

[55]  exhibit  changes  which  are  readily  followed  under  the  microscope.  It 
is  better  still  to  feed  the  Planarians  with  the  blood  of  those  lower 
Vertebrates  whose  red  corpuscles  are  nucleated.  In  my  researches 
I  have  used  the  blood  of  the  goose.  The  red  blood  corpuscles 
of  this  bird,  when  ingested  by  the  epithelial  cells  of  the  intestine 
of  Planarians,  are  usually  collected  into  compact  groups  (fig.  7), 
only  a  few  remaining  isolated.  The  majority  of  these  red  corpuscles 
soon  lose  their  normal  appearance  and  contour ;  they  become 


FIG.  6.  Young  Planarian 
some  time  after  having 
sucked  goose's  blood. 


Preliminary  remarks  on  immunity  in  animal  kingdom  51 


rounded   and  fused   together,  but   the  nucleus   and    the   haemo- 
globin enable  us  to  recognise  them  without  any  difficulty.    Later 


FIG.  7.  Intestinal  ceU  of  a 
Planarian,  filled  with  red 
blood  corpuscles,  undergoing 
digestion,  of  the  goose. 


FIG.  8.  Digestion  of  red  blood 
corpuscles  of  the  goose  with- 
in an  intestinal  cell  of  a 
Planarian. 


he  red  colouring  matter  begins  to  diffuse  into  the  digestive  vacuoles 
which  form  around  the  corpuscles.  These  corpuscles  empty  them- 
elves,  retaining  their  nuclei  and  capsules,  which  shrivel  more  and 
more.  The  nucleus  also  undergoes  almost  complete  digestion,  its 
membranous  layer  alone  persisting  (fig.  8).  Even  several  days  after 
;he  digestion  of  the  blood  has  begun  one  can  still  find  debris  of 
>erfectly  recognisable  red  corpuscles,  but  the  red  colour  has  been 
•eplaced  by  a  more  or  less  pronounced  brown  tint.  In  the  last 
stage  of  the  digestive  process,  as  the  red  corpuscles  disappear,  the 

4—2 


52 


Chapter  III 


protoplasm  of  the  intestinal  cells  becomes  filled  with  round  vacuoles, 
containing  brown  irregular  concretions— excreta— which  are  expelled 
into  the  intestinal  cavity. 

This  slow  digestion  of  a  substance  usually  so  easily  assimilable  as 
blood  takes  place  entirely  within  the  epithelial  cells  of  the  intestine. 
Continuous  microscopical  observation  demonstrates  most  clearly  the 
complete  absence  of  any  extracellular  digestion  of  the  blood  corpuscles 
in  the  intestinal  content. 

[56]  When  goose's  blood  mixed  with  blue  litmus  powder  is  given  to 
Planarians,  the  coloured  grains  may  be 
found  some  hours  afterwards  inside  the 
epithelial  cells  of  the  intestine,  but  only 
a  few  of  the  blue  litmus  granules  change 
colour,  taking  on  a  light  violet  tinge  ;  the 
great  majority  retain  their  blue  colora- 
tion. It  might  be  concluded  from  this 
that  in  Planarians  intracellular  digestion 
is  effected  in  a  neutral  or  nearly  neutral 
medium.  If,  however,  the  preparations 
of  intestinal  cells  gorged  with  goose's 
blood  are  treated  with  a  1%  solution 
of  neutral  red,  we  at  once  notice  that 
the  red  corpuscles  and  the  vacuoles 
which  contain  them  are  stained  bright 
red,  assuming  a  tint  similar  to  that  given 
with  picrocarmine  staining  (fig.  9).  This 
colour  reaction  indicates,  according  to 
our  researches  on  neutral  red,  an  acid  reaction,  more  feeble,  however, 
than  that  met  with  in  Paramaecium  and  many  other  Protozoa. 

Macerations  of  Planarians  in  normal  saline  solution  to  which  has 
been  added  a  small  quantity  of  the  red  corpuscles  of  the  goose's 
blood  exhibit  in  vitro  a  very  distinct  solvent  action  on  these  cor- 
puscles, which  become  rounded  and  lose  their  haemoglobin,  this  latter 
diffusing  into  the  surrounding  fluid,  and  at  the  close  of  the  experimen 
there  remain  simply  the  membranes  and  the  nuclei  of  the  corpuscles 
The  study  of  these  Planarians  shows  us,  then,  that  the  food  of  the 
animals  undergoes  exclusively  intracellular  digestion  in  a  feebly  acid 
medium  and  by  means  of  a  soluble  ferment,  and  it  furnishes  us  with 
proof  that  typical  intracellular  digestion  is  essentiallya  chemical  process 
due  to  the  intervention  of  enzymes.  Now  there  can  be  no  question,  here, 


FIG.  9.  Portion  of  an  intestinal 
cell  of  a  Planarian,  treated 
with  1%  neutral  red. 


Preliminary  remarks  on  immunity  in  animal  kingdom  53 

of  a  protoplasmic  action  proper,  but  the  branched  digestive  canal, 
so  intimately  associated  with  the  parenchyma,  cannot  be  completely 
isolated  from  the  rest  of  the  Planariau,  and  it  is  impossible  to  study 
in  vitro  its  digestive  action  apart  from  other  tissues.  To  attain  this 
end  we  must  turn  to  animals  of  larger  size  and  those  in  which  the 
digestive  organs  can  be  isolated  more  easily.  In  the  Coelenterata 
intracellular  digestion  is  general  Many  of  them  are  so  transparent 
that  they  can  be  examined  in  vivo.  It  is  easy  to  observe  that  the 
particles  of  food  are  seized  by  amoeboid  processes  of  the  entodermic 
cells  and  that  they  pass  into  the  substance  of  these  elements  there  to 
be  digested.  For  the  systematic  study  of  the  digestive  phenomena, 
however,  it  is  not  sufficient  merely  to  examine  all  that  takes  place  in 
the  living  animal.  Experiment  in  vitro  is  also  necessary.  For  this  [57] 
purpose  the  Actinians  or  sea-anemones  offer  us  really  excellent 
material.  As  these  animals  are  very  common  in  all  our  seas  and 
are  easily  kept  alive  for  long  periods  in  aquaria,  they  have  been  used 
for  various  researches,  among  others  for  the  study  of  the  process  of 
digestion. 

The  Actinians  are  easily  fed  in  captivity;  they  devour  morsels 
of  flesh,  of  shrimps,  of  mollusca  and  other  marine  animals  with 
avidity.  The  ingenious  English  observers  Couch  and  G.  H.  Lewes1 
long  ago  demonstrated  that  morsels  of  food  when  introduced  enclosed 
in  perforated  quills  or  wrapped  in  test  paper  or  gutta  percha  silk  and 
swallowed  by  the  anemones  were  afterwards  ejected  surrounded  by 
mucus  but  with  no  trace  of  digestion.  Having  failed  in  their  search 
for  digestive  juices  in  the  large  gastric  or  coelenteric  cavity  of  the 
Actinians,  Lewes  concluded  that  digestion  in  these  animals  is  effected 
in  a  purely  mechanical  fashion.  The  greatly  developed  muscles  of  the 
Actinians  were  supposed  to  squeeze  the  food  and  extract  its  fluid  which 
is  then  absorbed  by  the  walls  of  the  general  cavity.  It  was  not  until 
very  much  later  that  the  problem  of  digestion  in  the  Actinians  could 
be  resolved  in  any  accurate  and  definitive  fashion.  More  than  twenty 
years  ago  I  demonstrated2  that  the  digestion  in  these  polyps  is  intra- 
cellular. In  order  that  a  clear  conception  of  this  phenomenon  may  be 
obtained  it  may  be  useful  to  recall  in  a  few  words  the  fundamental 
features  of  the  organisation  of  Actiuiaus.  They  are  cylindrical  bodies, 
sometimes  as  large  as  the  fist,  attached  by  their  base  to  stones, 
shells,  or  other  submarine  objects,  and  furnished  at  their  free 
extremity  with  one  or  more  series  of  tentacles.  In  the  middle 

1  G.  H.  Lewes,  "Sea-side  Studies/'  Edin.  and  London,  1858,  p.  216. 

2  Zool.  Am.,  Leipzig,  1880,  Jahrg.  HI,  S.  261,  and  1882,  Jahrg.  v,  S.  310. 


54 


Chapter  III 


of  this  extremity  is  an  elongated  opening,  the  mouth,  which 
leads  into  a  spacious  sac,  often  spoken  of  as  the  stomach.  It  is, 
however,  only  a  kind  of  oesophagus,  through  which  the  food  passes 
into  the  large  coelenteric  cavity  which  is  divided  by  septa  into 
numerous  compartments  lined  by  the  entodermic  epithelium.  These 
septa  give  origin  to  many  very  long  and  tortuous  filaments,  spoken  of 
as  mesenterial  filaments  from  their  resemblance,  a  purely  superficial 
one,  to  the  mesentery  of  higher  animals  (fig.  10).  When  the  Actinian 
is  hungry  it  protrudes  its  tentacles  in  order  to  seize  marine  animals, 
which  it  conducts  to  its  mouth.  The  lips  and  the  oesophagus  are 
[58]  used  to  estimate  the  quality  of  the  capture,  and  if  it  is  found 
unsuitable  the  anemone  rejects  it,  first  surrounding  it  with  a  layer  of 


FIG.  10.     Longitudinal  section  of 
an  Actinian  (after  Hollard). 


FIG.  11.  An  Actinian  in  which  carmine 
after  absorption  has  passed  into  the 
mesenterial  filaments. 


mucus.  If  however  the  food  is  found  to  be  suitable,  the  Actinian 
retains  it  in  its  large  cavity  and  throws  around  it  a  multitude  of  its 
mesenterial  filaments.  These  penetrate  it  in  all  directions,  and  as 
their  epithelial  cells  are  capable  of  sending  out  amoeboid  processes 
they  seize  and  ingest  the  particles,  which  immediately  enter  the  proto- 
plasmic content.  This  work  is  done  with  such  precision  and  nicety 
that  the  sea-anemone  is  able  to  extract  the  contents  of  a  shrimp  from 
the  carapace,  which  latter  alone  it  rejects. 


Preliminary  remarks  on  immunity  in  animal  kingdom  55 

The  epithelium  of  the  mesenterial  filaments  is  therefore  the  organ 
of  digestion  in  the  Actinians.  The  nutritive  parts  of  their  prey  pass 
into  the  amoeboid  epithelial  cells  and  there  undergo  a  purely  intra- 
cellular  digestion.  If  we  add  to  the  shrimp-muscle  or  other  food 
a  little  carmine  or  blue  litmus  powder,  the  mesenterial  filaments 
ingest  it  also  and  become  pigmented.  After  eating  carmine  they 
assume  a  very  brilliant  rose  colour  (fig.  11) ;  blue  litmus  colours  them  [59] 
rose  violet.  This  change  of  colour  in  the  interior  of  the  cells  of  the 
filaments  indicates  a  decidedly  acid  reaction  of  their  contents1.  When 
one  adds  to  the  mesenterial  filaments  which  are  carrying  on  the  process 
of  digestion  a  drop  of  a  1  °/0  solution  of  neutral  red  they  assume  various 
shades  of  red  (fig.  12). 

This  intracellular  digestion  in  the  Actinians  has  been  confirmed 
by  several  observers,  amongst  whom  may 
be  cited  Chapeaux2  and  Bjelooussoff3. 
It  has  often  been  asserted,  however, 
that,  along  with  a  digestion  in  the  in- 
terior of  the  cells  of  the  mesenterial 
filaments,  there  is,  in  the  Actinians,  a 
secretion  in  the  coelenteric  cavity  of 

their  body  of  fluids  which  digest  nutri-  FIG.  12.  Portion  of  mesenterial 
tive  matter  by  means  of  a  soluble  filament  of  an  Actinian, 

ferment.    A  ferment  similar  to  trypsin  **ined  with  10''  neutral 

has  been  extracted  from  Actinians  by 

Leon  Fredericq  and  Krukenberg.  But,  in  presence  of  contradictory 
assertions,  it  remained  undecided  whether,  in  the  enzymatic  digestion, 
this  ferment  does  its  work  in  the  fluid  of  the  coelenteric  cavity  or 
whether  it  represents  the  active  factor  in  intracellular  digestion. 

With  the  object  of  definitely  elucidating  a  problem  of  such  general 
importance,  Mesnil,  the  superintendent  of  my  laboratory,  has  been 
good  enough  to  carry  out  a  fresh  series  of  experiments  on  the  digestion 
of  the  Actinians  and  has  studied  this  process  not  only  in  animals  kept 
in  captivity  in  aquaria  but  also  in  Actinians  living  under  natural  con- 
ditions in  the  sea4. 

As  intracellular  digestion  is  of  interest  to  us  specially  in  connection 
with  the  resorption  of  formed  elements  in  the  tissues  and  cavities  of 

1  Metchnikoff.  Ann.  de  TInst.  Pasteur,  Paris,  1893,  t.  vn,  p.  348. 

2  Bull.  Acad.  roy.  de  JBelg.,  Brux.,  1893,  t.  xxv,  p.  262,  and  Arch,  de  Zool  exper., 
Paris,  1893,  3me  serie,  t  I,  p.  139. 

3  "Etudes  de  physiologic  sur  les  Actinies,"  Charkoff,  1895  (in  Russian). 

4  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  352. 


56  Chapter  III 

animals,  Mesnil  directed  his  attention  to  the  digestion  of  the  red 
corpuscles  of  the  blood.    He  made  use  of  the  red  corpuscles   of 

[60]  several  species  of  Yertebrata,  but  he  made  a  special  study  of  the 
digestion  of  nucleated  red  blood  corpuscles.  These  corpuscles  are 
very  delicate,  and  may  even  undergo  a  certain  degree  of  maceration 
in  ordinary  sea  water.  In  spite  of  this  these  red  corpuscles  are  not 
digested  in  the  coelenteric  cavity  of  the  Actinians  but,  once  ingested 
by  the  entodermic  cells  of  the  mesenterial  filaments,  they  are  com- 
pletely dissolved  by  the  intracellular  digestion.  Mesnil  also  observed 
that  fibrin  is  not  digested  except  in  the  cells  of  the  filaments.  The 
facts  cited  by  Chapeaux  in  favour  of  an  extracellular  digestion  in  the 
fluid  of  the  coelenteric  cavity  in  no  way  support  his  hypothesis,  and 
reduce  themselves,  according  to  Mesnil,  to  a  digestion  by  the  diastase 
of  blood  itself  fixed  by  the  fibrin,  after  the  bleeding,  at  the  moment 
of  the  formation  of  the  clot. 

For  a  certain  period  the  red  corpuscles  may  be  met  with  inside  the 
cells  of  the  mesenterial  filaments.  They  are  ingested  in  their  normal 
state — oval  red  corpuscles  with  a  nucleus.  As  several  hours  are 
required  for  the  ingestion,  it  is  evident  that  the  fluid  of  the  coelenteric 
cavity  has  been  incapable  of  attacking  the  red  corpuscles.  In  the 
protoplasm  of  the  entodermic  cells  the  red  corpuscles  become  rounded, 
their  walls  become  permeable,  and  the  haemoglobin  begins  to  diffuse 
from  them.  It  passes  first  into  the  vacuoles  of  the  digestive  cells  and 
is  then,  in  part,  ejected  into  the  general  body  cavity.  The  haemo- 
globin is  transformed  into  a  green  substance  which  reminds  one  of 
biliary  pigment.  The  membranes  and  nuclei  of  the  red  corpuscles 
are  also  digested  and  ultimately  disappear  completely. 

The  digestive  cells  of  the  entoderm  ingest  not  only  blood  cor- 
puscles or  fibrin,  but  also  fragments  of  muscular  fibre  and  particles  of 
carmine  and  litmus.  These  latter,  as  already  stated,  indicate  a  marked 
acid  reaction. 

In  the  Actinians,  then,  the  mesenterial  filaments,  or  rather  their 
entodermic  portion,  represent  the  real  organ  of  intracellular  digestion. 
There  are  indeed  other  regions  of  the  entoderm  which  also  carry  on 
this  function,  but  in  an  insignificant  degree  as  compared  with  the 
mesenterial  filaments  which  are  capable,  however,  not  only  of 
ingesting  and  digesting  solid  substances,  but  also  of  absorbing 
solutions.  Mesnil  has  demonstrated  this  by  injecting  soluble 

[6i]  colouring  matters,  such  as  eosin,  carminate  of  ammonia,  etc.,  into 
Actinians.  These  solutions,  although  in  great  part  absorbed  by  the 


Preliminary  remarks  on  immunity  in  animal  kingdom  57 

digestive  cells  of  the  mesenterial  filaments,  can,  however,  also  be 
retained  by  other  elements,  amongst  others,  the  cells  of  the  ectoderm. 

As  the  digestion  of  the  food-particles  goes  on  within  the  ento- 
dermic  cells  of  the  mesenterial  filaments  and  as  these  organs  can 
easily  be  isolated  from  the  rest  of  the  Actinian,  Mesnil  was  able  to 
study  with  great  precision  and  care  the  phenomena  of  digestion 
outside  the  organism.  "With  this  object  he  prepared  extracts  of  the 
filaments  in  sea-water  and  studied  their  action  on  various  nutritive 
substances.  He  confirmed  the  discovery  of  a  soluble  ferment  made 
by  Leon  Fredericq  and  demonstrated  that  it  is  capable  of  digesting 
albuminoid  substances  (fibrin,  coagulated  albumen)  in  media  which 
are  neutral,  slightly  alkaline  or  weakly  acid.  In  this  respect  the 
actino-diastase  (the  name  given  by  Mesnil  to  the  soluble  ferment 
of  the  Actinians)  approaches  most  nearly  to  papain.  On  the  other 
hand,  it  is  distinguished  by  its  greater  sensitiveness  to  an  excess 
of  acid  and  also  by  its  more  powerful  action  on  coagulated  albumen. 

The  actino-diastase  acts  vigorously  at  any  temperature  between 
15°  and  20°  C.,  but  the  optimum  temperature  for  its  digestive  action 
is  between  36°  and  45°  C.  Higher  temperatures  weaken  the  diastatic 
power,  and  heating  to  55 — 60°  C.  inhibits  it  completely.  Among  the 
products  of  the  digestion  of  albuminoids  by  actino-diastase,  Mesnil, 
like  his  predecessors,  found  not  only  a  notable  quantity  of  peptone 
but  also  products  of  the  disintegration  of  the  albuminoid  molecule, 
such  as  tyrosin  and  proteino-chromogeu.  Consequently  actino- 
diastase  resembles  Mouton's  amoebo-diastase  in  certain  respects. 

The  nucleated  red  blood  corpuscles  of  the  lower  Yertebrata  are 
very  convenient  objects  on  which  to  observe  the  process  of  intracellular 
digestion  within  the  cells  of  the  mesenterial  filaments.  Mesnil  has  also 
studied  them  in  vitro  under  the  influence  of  actino-diastase.  Under 
these  conditions  the  phenomena  of  digestion  recall  very  clearly  those 
that  have  been  observed  within  the  digestive  cells.  The  oval  red 
corpuscles  of  the  fowl  and  goose  become  spherical  as  a  result  of  the 
solvent  action  on  their  membrane,  and  the  haemoglobin  diffuses  into 
the  fluid.  The  membranes  and  the  nuclei  of  the  corpuscles  are,  how- 
ever, little  altered  and  may  be  recognised  under  the  microscope.  The 
difference  between  this  and  digestion  within  the  cells  reduces  itself 
to  a  more  feeble  digestive  action  of  the  aqueous  extract.  It  is  evident 
that  the  preparation  of  this  extract  is  only  capable  of  bringing  into  [62] 
prominence  a  certain  proportion  of  the  actino-diastase  contained  in 
the  entodermic  cells  of  the  filaments. 


58  Chapter  III 

Mesnil  has  fed  the  same  Actinians  with  repeated  doses  of  blood 
with  a  view  to  make  out  whether  the  cells,  tinder  these  conditions, 
acquire  any  special  aptitude  for  the  production  of  the  actino-diastase, 
Notwithstanding  numerous  attempts,  he  could  never  assure  himself 
that  this  takes  place  ;  the  rapidity  with  which  the  red  corpuscles  were 
dissolved  by  the  extract  of  the  mesenterial  filaments  was  the  same 
whether  this  was  prepared  from  Actinians  that  had  been  several  times 
fed  on  blood  or  from  those  that  had  received  none  at  all. 

From  what  I  have  just  described  no  doubt  can  exist  that  intra- 
cellular  digestion  is  not  a  " protoplasmic"  process  essentially  different 
from  that  which  is  brought  about  by  the  digestive  juices  secreted  in 
the  intestinal  canal.  In  both  cases  we  have  a  diastatic  action,  due 
to  soluble  ferments,  produced  by  living  elements.  In  intracellular 
digestion,  however,  the  diastases  carry  on  digestion  in  the  interior 
of  the  cells,  principally  in  the  vacuoles,  whilst  in  extracellular 
digestion  this  process  goes  on  outside  the  cells,  in  the  lumen  of 
the  gastro-intestinal  canal. 

It  cannot  be  doubted  that,  in  the  animal  scale,  intracellular 
digestion  represents  an  earlier  and  primitive  condition  for  the 
solution  of  the  food  substances.  This  follows  from  the  fact  that 
it  is  widely  distributed  amongst  the  lowest  animals,  such  as  the 
Protozoa,  Sponges,  Coelenterata  and  Turbellaria.  Intracellular 
digestion  only  gives  way  step  by  step  to  digestion  by  secreted 
juices.  The  higher  Invertebrata  furnish  us  with  conclusive  testi- 
mony on  this  point.  Thus,  among  the  gasteropod  Mollusca,  there  are 
some  which  exhibit  the  two  modes  of  digestion  in  the  same  animal. 
In  Phyllirhoe,  a  beautiful  mollusk,  without  a  shell  and  quite  trans- 
parent, which  floats  on  the  surface  of  the  sea,  the  food  can  be  seen 
passing  into  the  cavity  of  the  digestive  canal,  where  it  undergoes 
a  preliminary  digestion  by  secreted  juices  ;  the  result  is  a  magma  of 
small  solid  particles  which  are  at  once  seized  by  the  amoeboid 
epithelium  of  the  coecal  appendages,  two  on  each  side  of  the  body. 
Intracellular  digestion  then  completes  the  process  and  ends  by 
dissolving  the  nutritive  substances  and  reducing  them  to  their 
final  stage  previous  to  absorption.  On  adding  'to  the  food  some 
particles  of  carmine  these  may  be  found  along  with  the  digestible 
particles  in  the  interior  of  the  epithelial  cells  of  the  coeca. 
[63]  This  example  furnishes  us  with  a  real  link  between  primitive 
intracellular  digestion  and  the  perfected  and  derivative  extracellular 
digestion.  In  the  same  group  of  Gasteropods  may  be  followed  out 


Preliminary  remarks  on  immunity  m  animal  kingdom  59 

several  stages  of  this  evolution  so  that  in  the  higher  representatives  of 
the  group,  such  as  the  slugs  and  the  snails,  we  meet  with  digestion 
carried  on  only  by  secreted  juices  in  the  gastro-intestinal  contents. 
In  these  Mollusca  a  voluminous  glandular  organ,  the  liver,  which  is  . 
certainly  derived  from  coecal  appendices  similar  to  those  of  Phyllirhoe, 
is  now  met  with.  Regarded  from  this  point  of  view  the  liver  is,  as 
Claude  Bernard  has  stated,  an  organ  of  second  digestion.  I  think 
that  a  detailed  study  of  the  liver  of  the  Mollusca,  guided  by  this  idea, 
will  give  results  of  considerable  importance. 

In  the  Vertebrata  intracellular  digestion  in  the  gastro-intestinal 
canal  almost  disappears  and  is  replaced  by  digestion  carried  on 
by  means  of  ferments  contained  in  secreted  juices.  We  cannot,  of 
course,  offer  to  the  reader  anything  like  a  complete  account  of 
this  extracellular  digestion  in  the  higher  animals.  It  is  necessary, 
however,  to  draw  attention  to  several  aspects  of  this  function  which 
have  been  established,  thanks  to  the  progress  made  during  recent 
years,  in  obtaining  digestive  juices  and  in  the  study  of  their  action. 

For  the  study  of  intracellular  digestion  the  sea-anemone  is  the 
most  suitable  animal  for  our  purpose ;  for  that  of  extracellular 
digestion  the  dog.  In  this  latter  animal,  an  omnivorous  flesh-eater, 
the  food-substances  are  treated  by  digestive  juices  of  great  activity 
which  contain  a  whole  series  of  soluble  ferments.  The  stomach 
secretes  two  of  these :  rennet  and  pepsin.  The  pancreas  elaborates 
three  :  trypsin,  amylase  and  saponase,  which  act  on  the  three  main 
groups  of  food-substances.  To  these  the  small  intestine  adds  a  special 
ferment,  described  by  Pawloff1  under  the  name  of  enterokynase. 
Every  one  recognises  the  proteolytic  function  of  pepsin  and  trypsin 
and  the  analogies  and  differences  between  these  two  diastases.  Nor 
need  I  dwell  on  amylase  or  on  the  ferment  which  saponifies  fats.  But 
enterokynase  merits  special  attention  in  connection  with  the  study  of 
immunity.  Pawloff  entrusted  to  his  pupil  Che"powalnikoff  the  study 
of  the  digestive  role  of  the  intestinal  juice  concerning  which,  up  to 
this,  very  little  was  known.  It  was  known  indeed  that  this  juice 
contained  weak  saccharifying  and  inverting  ferments,  but  it  was  [64] 
generally  regarded  as  a  secretion  of  little  importance.  Chepowal- 
nikoff2  has  demonstrated  that  this  view  is  absolutely  erroneous.  The 
intestinal  juice  fulfils  the  very  important  function  of  accelerating  the 

1  Address  delivered  before  the  Societe  des  medecins  russes  at  St  Petersburg. 
Gaz.  din.  de  Botkine,  1900. 

2  "  Physiologic  du  sue  intestinal,"  Saint-Petersbourg,  1899  (Thesis,  in  Russian). 


60  Chapter  111 

action  of  the  three  pancreatic  ferments.  The  duodenal  juice  of  the 
dog,  especially,  contains  enterokynase.  When  this  juice  is  mixed  with 
a  pancreatic  juice  that  by  itself  actively  digests  fibrin  and  albumen, 
digestion  takes  place  still  more  rapidly,  the  action  being  from  three 
to  four  times  as  great.  The  part  played  by  the  intestinal  juice 
becomes  even  more  evident  when  it  is  mixed  with  a  pancreatic  juice 
that  has  little  or  almost  no  activity,  as  is  the  case  of  that  from  dogs 
that  have  recently  been  operated  upon.  Thus  pancreatic  juice,  which 
has  no  action  upon  albumen,  digests  it  promptly  when  a  certain 
quantity  of  duodenal  juice  is  added.  When  Che*powalmkoff  took 
500  c.c.  of  inactive  pancreatic  juice  diluted  with  500  c.c.  of  water 
or  soda  solution  and  added  to  it  but  a  single  drop  of  intestinal  juice, 
the  mixture  exerted  a  manifest  digestive  action  on  coagulated 
albumen. 

If,  in  place  of  pancreatic  juice,  we  take  the  aqueous  or  glycerinated 
extract  of  the  pancreas,  which  by  itself  exerts  a  very  insignificant 
digestive  action  on  albumen,  and  add  to  it  intestinal  juice,  digestion 
takes  place  immediately.  If  it  be  admitted,  as  several  physiologists 
maintain,  that  the  inactivity  of  the  pancreas  is  due  to  the  fact  that  we 
have  zymogen  present  in  place  of  trypsin,  one  might  conclude  with 
Chepowalnikoff  that  "the  intestinal  juice  possesses  the  power  of 
transforming  the  zymogen  into  trypsin,  and  that  this  transformation 
takes  place  in  a  much  more  marked  degree  than  in  the  presence 
of  acids  or  the  oxygen  of  the  air"  (p.  137). 

The  intestinal  juice,  from  whatever  region  of  the  small  intestine  it 
be  derived,  exercises  an  undoubtedly  favourable  influence  on  the 
digestion  of  starch  by  the  pancreatic  juice,  but  this  action  is  much 
more  feeble  than  that  on  trypsin  digestion.  The  action  of  the 
intestinal  juice  on  the  saponification  of  fats  is  even  less  marked. 
But  here  it  is  to  the  bile  that  the  more  important  rdle  is  transferred. 
This  fluid  also  augments  the  activity  of  the  pancreatic  juice,  but 
in  a  manner  different  from  the  intestinal  juice,  for  it  acts  especially 
by  accelerating  the  digestion  of  fatty  substances. 

[65]  The  action  on  the  pancreatic  digestion  is  not  in  any  way  interfered 
with  when  the  bile  is  heated  to  boiling  point.  On  the  other  hand 
the  intestinal  juice,  under  these  conditions,  completely  loses  its 
accelerating  role.  It  follows  from  this,  as  has  been  formulated  by 
Pawloff,  that,  in  the  intestinal  juice,  the  existence  of  a  soluble  ferment 
which  is  destroyed  by  heat  must  be  admitted;  to  this  ferment  he 
proposes  to  give  the  name  of  enterokynase.  Without  exercising 


Preliminary  remarks  on  immunity  in  animal  kingdom  61 

a  digestive  power  on  any  of  the  alimentary  substances,  it  may  act  as 
a  ferment  of  the  pancreatic  ferments. 

Delezenne,  at  the  Pasteur  Institute,  has  repeated  ChepowalnikofFs 
experiments.  He  has  confirmed  the  accuracy  of  his  results  and 
has  added  new  data  of  great  importance,  not  only  as  regards  the 
physiology  of  digestion  but  also  in  relation  to  the  study  of  immunity. 
Enterokynase  appears  from  Delezenne's  experiments  to  be  a  true 
ferment ;  carried  down  by  the  same  precipitants  (collodion,  phosphate 
of  lime,  alcohol)  which  enable  us  to  obtain  the  greater  number  of  the 
known  ferments ;  it  is  sensitive  to  high  temperatures,  and  even  that  of 
65°  C.  is  sufficient  to  do  away  with  the  greater  part  of  its  activity. 
Yet  another  property  of  enterokynase,  which  it  possesses  in  common 
with  the  soluble  ferments  and  which  has  for  us  a  very  special  interest, 
is  the  facility  with  which  it  attaches  itself  to  fibrin.  By  means  of  flakes 
of  this  substance  we  can  at  any  time  remove  from  a  fluid  the  whole 
of  the  enterokynase  contained  therein.  This  fixative  property  is  very 
important  in  connection  with  the  part  which  enterokynase  plays  in 
digestion.  The  fibrin  to  which  it  has  become  attached  absorbs  trypsin 
with  great  avidity.  If  we  introduce  flakes  of  fibrin  impregnated  with 
enterokynase  along  with  other  flakes  which  have  not  been  in  contact 
with  this  ferment  into  a  solution  of  trypsin,  the  former  are  digested 
with  great  rapidity,  whilst  the  latter  do  not  undergo  any  change.  The 
fibrin  that  has  fixed  enterokynase  is  capable  of  clearing  a  fluid  of  its 
trypsin.  On  the  other  hand,  that  which  has  not  been  acted  upon  by 
the  intestinal  juice  leaves  it  there  almost  unaltered. 

It  is  of  the  utmost  importance  that  we  should  inform  ourselves  as 
to  the  origin  of  the  enterokynase  of  the  intestinal  fluid.  This  fluid, 
when  obtained  from  a  fistulous  opening,  for  example,  contains  mucus 
and  a  considerable  amount  of  debris  of  various  kinds  of  cells.  What 
are  the  elements  which  furnish  such  a  remarkable  ferment  ?  Dele-  [66] 
zenne  has  obtained  a  very  precise  answer  to  this  question.  The 
enterokynase  is  not  contained  in  the  mucus  and  is  not  secreted  by  the 
intestinal  glands  ;  it  comes  from  the  lymphoid  organs. 

If  the  small  intestine  of  a  fasting  dog  be  washed  carefully  with 
water  all  the  pre-existing  enterokynase  is  removed  from  it.  The 
Fever's  patches  are  then  removed  and  treated  with  chloroform 
water.  The  other  parts  of  the  small  intestine  are  similarly  treated. 
This  fluid  dissolves  the  enterokynase,  as  it  does  the  other  soluble 
ferments.  We  find  that  the  Peyer's  patches  furnish  enterokynase, 
but  that  the  rest  of  the  intestine,  including  Lieberkiihn's  glands, 
give  none. 


(52  Chapter  III 

We  know  that  the  Peyer's  patches  are  lymphoid  organs  in  which 
are  a  large  number  of  amoeboid  mononucleated  cells,  and  that  these 
elements  are  even  capable  of  ingesting  foreign  bodies  and  of  sub- 
mitting them  to  intracellular  digestion.  It  is  therefore  not  at  all 
astonishing  that  Delezenne  should  have  succeeded  in  finding  entero- 
kynase  in  the  mesenteric  glands  of  several  Mammals  (dog,  pig,  rabbit). 
These  glands,  when  treated  by  the  method  just  mentioned,  yield  a 
substance  which  assists  the  action  of  trypsin  just  as  does  the  intestinal 
juice.  Having  reached  this  point,  Delezenne  asked  himself  whether 
the  mononucleated  white  corpuscles,  so  closely  allied  to  the  mono- 
nucleated  cells  of  the  lymphoid  organs,  may  not  also  contain 
enterokynase.  With  the  object  of  settling  this  point  he  collected 
exudates  that  were  rich  in  mononucleated  leucocytes  ;  in  these  also 
he  found  this  same  soluble  ferment.  Moreover,  the  leucocytic  layer 
of  the  blood  showed  itself  equally  capable  of  increasing,  very 
energetically,  the  action  of  trypsin. 

The  results  of  the  old  experiments  carried  out  by  Schiff  and  by 
Herzen  on  the  adjuvant  rdle  of  the  extract  of  the  spleen  in  pancreatic 
digestion,  must  without  doubt  be  ranged  alongside  those  we  have  just 
indicated.  In  fact  the  mononucleated  cells  of  the  spleen,  like  those  of 
Peyer's  patches  and  of  the  mesenteric  glands,  contain  a  substance 
which  acts  like  enterokynase.  Delezenne  has  given  us  a  definite 
demonstration  of  its  presence  and  action. 

In  intracellular  digestion  it  is  the  chemical  side  which  has  been 
most  difficult  of  demonstration.  The  purely  physiological  functioning, 
the  sensitiveness  of  the  digestive  cells  and  the  amoeboid  movements 
[67]  of  their  protoplasmic  processes  are,  on  the  other  hand,  so  manifest 
that  it  has  even  been  suggested  that  intracellular  digestion  should  be 
looked  upon  as  a  protoplasmic  phenomenon  purely  vital  in  character. 

In  extracellular  digestion  through  the  agency  of  secreted  juices 
we  have  a  very  different  condition.  Here  the  chemical  side  is  the 
striking  feature,  the  physiological  factor  being  veiled  more  or  less 
completely.  Nevertheless,  thanks  to  recent  advances  and  above  all 
to  the  labours  of  Pawloff's  disciples  in  St  Petersburg,  this  problem 
has  been  elucidated  in  a  very  remarkable  fashion. 

The  secretion  of  digestive  fluids  follows  definite  laws,  the  most 
potent  factor  being  the  reflex  action  of  the  nervous  system.  To  use  the 
expression  of  Pawloff,  the  study  of  the  process  of  salivary  secretion 
has  revealed  a  real  psychology  of  these  organs.  You  may  fill  the 
mouth  of  a  dog  with  small  polished  pebbles  or  with  snow  ;  you  may 
pour  into  it  very  cold  water — the  saliva  will  not  flow.  But  merely 


Preliminary  remarks  on  immunity  in  animal  kingdom  63 

allow  the  animal  to  see  sand  in  the  distance — the  glands  at  once 
begin  to  secrete  fluid  saliva.  Tempt  the  dog  with  flesh — and 
immediately  a  thick  saliva  appears ;  show  him  dry  bread — saliva  is 
secreted  in  abundance,  even  if  the  dog  has  no  great  desire  to  eat 

The  same  phenomena  may  be  observed  in  the  stomach.  Mechanical 
stimulation  by  inert  bodies,  such  as  stones,  provokes  no  secretion ; 
but  the  suggestion  of  a  meal  or  the  sight  of  food  is  sufficient  to  call 
forth  a  large  quantity  of  gastric  juice.  The  quantity  and  quality  of 
the  gastric  juice  are  regulated  by  the  quantity  and  quality  of  the  food. 
Bread  given  to  a  dog  provokes  the  secretion  of  a  gastric  juice  endowed 
with  the  greatest  digestive  power.  That  which  flows  after  the  ingestion 
of  milk  contains  only  one- fourth  as  much  pepsin. 

In  spite  of  these  differences  in  the  gastric  secretion  in  relation 
to  food,  Pawloff  and  his  pupils  have  never  been  able  to  assure 
themselves  that  there  was  any  prolonged  and  chronic  adaptation  of 
the  gastric  function.  They  were  struck  by  the  uniformity  of  the 
digestive  power  of  a  great  number  of  their  dogs.  Samoiloff1  had 
under  observation  three  dogs  placed  on  different  diets.  In  spite  of 
the  very  long  periods  during  which  these  diets  were  given,  the  gastric 
juice,  in  all  the  dogs,  presented  the  same  properties  and  manifested 
no  appreciable  difference.  This  result  harmonises  with  that  indicated 
above  as  obtained  in  the  Actinians  fed  with  blood  by  Mesnil.  In  spite 
of  repeated  feedings  on  blood  from  the  same  species  of  animal,  the 
extract  from  the  meseuterial  filaments  was  in  no  way  different  from  [68] 
that  of  the  fasting  Actinians  used  for  control. 

The  pancreatic  secretion  is,  in  many  respects,  a  more  perfect  type. 
We  have  here  to  do  with  the  principal  agent  in  the  digestive  function, 
without  which  the  organism  could  not  continue  to  exist.  The  advances 
made  in  surgery  have  enabled  us  to  remove  the  stomach,  first  in  the 
dog  and  then  in  man,  and  there  are  already  several  persons2  from 
whom  the  stomach  has  been  removed  and  who,  in  spite  of  this 
operation,  have  continued  to  live.  A  portion  of  the  small  intestine 
may  also  be  removed,  but,  in  order  that  life  may  not  be  endangered, 
a  considerable  portion  of  it  must  be  left  intact.  It  is  evident  then 
that  the  pancreatic  digestion  is  an  admirably  organised  function  both 
in  animals  and  in  man.  One  of  the  main  regulators  of  this  process  of 
digestion  consists  in  the  great  sensitiveness  of  the  intestinal  mucous 
membrane.  Just  as  the  organs  of  the  buccal  cavity  possess  in  the 

1  Arch.  d.  sc.  liol,  St.-Petersb.,  1893,  t.  n,  p.  698. 

2  Of.  Bull.  Acad.  de  med.,  Paris,  1901,  p.  17. 


64  Chapter  III 

specific  sense  of  taste  an  excellent  means  of  discrimination  in  the 
choice  of  foods,  so  the  mucous  membrane  of  the  small  intestine  is 
endowed  with  a  special  sensitiveness,  comparable  to  the  chemiotaxis 
of  unicellular  organisms  and  of  the  cells  of  more  highly  developed 
organisms.  Hirsch  and  Mehring  have  satisfied  themselves  that  the 
passage  of  the  contents  of  the  stomach  through  the  pyloric  orifice 
depends  on  a  reflex  mechanism  which  proceeds  from  the  upper 
reaches  of  the  small  intestine.  To  the  researches  of  the  school  of 
Pawloff,  however,  we  owe  what  light  has  been  thrown  on  this  question. 
The  duodenal  mucous  membrane  is  endowed  with  a  well-developed 
chemiotaxis  for  acid  substances.  The  passage  of  the  acid  content 
of  the  stomach  into  the  duodenum  determines  this  chemiotaxis  and 
brings  about  a  secretion  of  alkaline  juice  which  neutralises  the  acid. 
This  contest  between  acid  and  alkali  forcibly  calls  to  our  mind  the 
analogous  phenomena  in  those  plants  that  defend  themselves  against 
the  alkaline  secretions  of  parasites  by  the  production  of  an  acid  (see 
Chapter  II).  As  in  these  lower  organisms,  this  battle  of  the  chemical 
secretions  is  regulated  by  the  action  of  living  and  sensitive  parts. 

When  the  acidity  of  the  mass  which  passes  through  the  pylorus  is 
too  marked,  the  reflex  contraction  starting  from  the  duodenal  mucosa 
arrests  its  passage.  Then  takes  place  a  neutralisation  of  the  acid, 
thanks  to  the  alkaline  secretion,  and  the  pylorus  is  again  allowed  to 
open.  This  mechanism  thus  regulates  the  passage  of  the  contents 
of  the  stomach  into  the  duodenum,  the  passage  taking  place  in 
instalments. 

[69]  The  sensitive  intestinal  mucous  membrane  can  estimate  not  only 
the  degree  of  acidity,  but  also  the  other  chemical  characters  of  the 
aliments  which  pass  into  the  duodenum.  This  chemiotaxis  is,  as  it 
were,  the  starting  point  of  the  reflex  action  which  excites  the  pan- 
creatic secretion  with  its  contained  three  ferments.  The  passage  of 
bread  through  the  pylorus  excites  the  secretion  of  a  juice  very  rich  in 
amylase  and  very  poor  in  saponase.  The  passage  of  milk  into  the 
duodenum  brings  forth,  on  the  other  hand,  a  juice  very  much  richer 
in  saponase  but  poorer  in  amylase  and  in  trypsin.  Flesh-meat 
provokes  the  secretion  of  a  pancreatic  juice  which  is  less  rich  in 
amylase  than  the  juice  poured  on  bread,  but  richer  In  saponase.  Fat 
causes  the  secretion  of  a  juice  still  richer  in  saponase  than  is  the  juice 
poured  out  in  the  presence  of  bread  or  milk.  These  facts  now 
carefully  established— especially  by  Walter1— demonstrate  that  the 
1  Arch.  d.  sc.  biol.,  St.-Petcrsb.,  1899,  t.  VII,  p.  1. 


Preliminary  remarks  on  immunity  in  animal  kingdom    65 

pancreatic  function  is  carefully  regulated  as  regards  its  adaptation  to 
the  characters  of  the  food  substances  on  which  it  is  to  act  Such 
adaptation  may  even  become  permanent. 

Whilst,  as  already  stated,  the  stomach,  under  the  influence  of  a  fixed 
diet,  is  incapable  of  effecting  any  lasting  modification  in  the  composition 
of  its  secreted  juice,  the  pancreas  may  reach  this  degree  of  perfection. 
When  a  dog  is  fed  for  several  weeks  on  bread  or  on  milk  and  is  then 
placed  on  flesh  diet  its  pancreatic  juice  is  found  to  become  progres- 
sively richer  in  trypsin.  Whilst  this  augmentation  of  the  proteolytic 
power  is  being  brought  about,  the  juice  becomes  poorer  and  poorer  in 
amylase.  Wassilieff x  has  carried  out  a  large  number  of  experiments 
on  this  point  and  has  demonstrated  a  very  remarkable  adaptation 
of  the  pancreatic  juice  to  the  wants  of  nutrition,  an  adaptation  that 
may  become  permanent.  A  dog  which  has  been  accustomed  to  digest 
bread  and  milk  adapts  itself  to  this  nourishment :  its  pancreatic  juice 
contains  less  and  less  trypsin,  but,  on  the  other  hand,  becomes  richer 
in  amylase.  Pawloff  observed  that  in  dogs  great  variations  in  the 
composition  of  the  pancreatic  juice  are  often  present ;  this  he  attributes 
to  the  diet  to  which  these  animals  had  been  previously  subjected. 

Not  only  does  the  quality  of  the  digestive  juices  accommodate  itself 
to  the  wants  of  digestion ;  their  quantity  also  undergoes  variations 
according  to  the  part  that  these  juices  have  to  play.  Thus,  Pawloff 
has  observed  that  his  dogs  secreted  a  saliva  which  was  very  fluid  and 
very  abundant  when  he  gave  them  acids,  bitter  substances  or  other  sub-  [70] 
stances  they  did  not  like.  On  the  other  hand,  the  presence  of  food  in 
the  mouth,  or  even  the  sight  of  it,  excited  the  secretion  of  a  thick  saliva 
containing  a  large  quantity  of  mucin.  In  the  first  case  the  part  played 
by  the  saliva  was  that  of  diluting  the  injurious  substances  as  much  as 
possible,  in  the  second  that  of  facilitating  the  deglutition  of  the  food. 

In  general  the  organism  manifests  a  tendency  to  produce  more 
digestive  ferments  than  it  actually  needs  for  digestion.  It  is  for  this 
reason  probably  that  they  are  often  found  outside  the  digestive  canal. 
Among  these  ferments  pepsin  and  amylase,  especially,  have  been 
definitely  proved  to  be  present  in  the  urine  of  man  and  of  some 
mammals,  notably  the  dog.  The  data  as  to  rennet  and  trypsin  are 
not  so  well  established.  But,  as  several  of  these  ferments,  such  as 
amylase  and  trypsin,  may  be  derived  from  several  sources  in  the 
organism,  their  elimination  by  the  urine  is  less  important  for  the 
thesis  I  have  just  formulated  than  is  that  of  pepsin. 

1  Arch.  d.  sc.  biol.,  St.-Petersb.,  1893,  t.  n,  p.  219. 
B.  5 


66  Chapter  III 

Pepsin  was  found  in  the  urine  by  Briicke  exactly  forty  years  ago. 
It  is  more  frequently  found  in  the  morning  urine,  but  is  absent  from 
that  passed  immediately  after  the  principal  meal.  Leo  and  Senator1 
found  only  traces  of  pepsin  during  the  prolonged  fast  of  the  Italian 
Cetti ;  but  the  day  he  broke  his  fast  they  were  able  to  demonstrate 
the  presence  of  a  considerable  quantity  of  this  ferment  in  his  urine. 

Delezenne  and  Froin,  with  the  object  of  seeking  the  source  of  the 
urinary  pepsin,  extirpated  the  stomach  of  a  dog.  After  the  animal 
had  recovered,  they  fed  it  well  and  examined  its  urine  at  different 
periods  of  the  day.  By  the  methods  which  had  shown  the  presence  of 
pepsin  in  all  the  normal  dogs  taken  as  controls  they  could  never 
discover  the  faintest  trace  of  this  diastase  in  the  urine  of  the  dog  that 
had  been  operated  upon.  On  the  other  hand,  the  urine  of  a  dog 
whose  stomach  had  simply  been  isolated,  contained  very  much  the  same 
quantity  of  pepsin  as  that  of  normal  dogs.  This  experiment  proved 
among  other  things  that  the  pepsin,  before  it  could  be  eliminated  by 
the  kidneys,  must  have  been  re-absorbed  by  the  wall  of  the  stomach. 
[71]  From  these  data,  combined,  it  must  therefore  be  admitted  that  the 
pepsin  found  in  the  blood  and  which  passes  thence  into  the  urine  can 
only  be  of  gastric  origin.  As  it  serves  no  useful  purpose  in  the  organ- 
ism we  must  conclude  that  a  portion  of  the  pepsin,  secreted  by  the 
stomach  and  not  used  for  digestion,  has  been  rejected  as  superfluous. 

The  study  of  the  digestive  function  of  animals  gives  us  information 
on  a  large  number  of  points  of  the  highest  importance  for  the  com- 
prehension of  immunity.  Intracellular  digestion,  a  function  so  widely 
distributed  in  the  lower  animals,  is  very  intimately  connected  with  the 
phenomena  which  are  observed  when  micro-organisms  are  destroyed  in 
the  animal  organism.  Extracellular  digestion  furnishes  us  with  informa- 
tion concerning  many  of  the  features  of  progressive  adaptation,  similar 
to  those  which  are  observed  in  connection  with  acquired  immunity. 

When  we  examine  the  phenomena  of  intracellular  digestion  and 
those  of  secretory  digestion  as  a  whole,  we  see  that,  in  both,  the 
chemical  processes  are  subjected  to  the  influence  of  the  living  parts 
of  the  organism.  In  the  lower  animals,  it  is  the  protoplasm  of  the 
amoeboid  cells  which  regulates  the  chemical  processes  in  digestion ; 
in  the  higher  animals,  this  role  is  taken  by  a  very  complicated 
apparatus,  in  which  the  nervous  system  plays  a  predominant  part. 

1  Firchow's  Archiv,  1893,  Suppl.  to  Bd.  cxxxi,  S.  142.  The  question  of  urinary 
ferments  is  summarised  in  Keubauer  u,  VogePs  "Analyse  des  Harns,"  Wiesbaden, 
10to  Aufl.,  1898,  S.  599. 


CHAPTER    IV  [72] 

RESORPTION  OF  THE  FORMED  ELEMENTS 

Digestion  in  the  tissues. — Resorption  of  cells  in  the  Invertebrata. — Resorption  of  red 
corpuscles  by  the  phagocytes  of  the  Vertebrata. — Phagocytes. — Various  cate- 
gories of  these  cells. — Macrophages  and  microphages. — Part  played  by  macro- 
phages  in  the  resorption  of  the  formed  elements. — Digestive  property  of  the 
macrophagic  organs. — Solution  of  the  red  blood  corpuscles  by  the  blood 
serums. — The  two  substances  which  operate  in  haemolysis.  Macrocytase  and 
fixative. — Analogy  of  the  latter  with  enterokynase. — Escape  of  the  macrocytase 
during  phagolysis.  Suppression  of  phagolysis.  Resorption  of  the  spermatozoa. — 
Presence  of  fixatives  in  plasmas. — Origin  of  fixatives. 

IT  is  usually  understood  that  nutritive  substances  must  necessarily 
be  subjected  to  the  influence  of  the  digestive  juices  in  the  gastro- 
intestinal canal  before  they  can  be  utilised  for  the  nutrition  of 
the  organism.  This  is  a  very  old  idea.  It  was  based  on  a  well- 
known  experiment  by  Schiff  who  injected  several  animals  intra- 
venously Avith  solutions  of  cane  sugar  and  egg  albumen  and  others 
with  the  same  substances  after  they  had  been  artificially  digested.  In 
the  first  case  the  food  substances  passed  into  the  urine,  in  the  second 
they  only  appeared  there  when  injected  in  large  quantities. 

At  the  recent  International  Congress  of  Medicine  held  in  Paris  in 
1900,  the  question  of  extra-buccal  nutrition  was  much  discussed1.  It 
has  been  accepted  that  fats,  when  injected  into  the  subcutaneous 
tissues,  are,  at  least  in  part,  absorbed  by  the  organism,  but  that 
carbo-hydrates  and  albuminoids  are  never  absorbed.  This  is  perhaps 
true  from  the  point  of  view  of  clinical  medicine.  But,  in  principle,  it 
must  be  admitted  that  food  substances  of  very  diverse  natures,  when 
introduced  into  the  organism  by  channels  other  than  the  gastro-[73] 
intestinal  canal,  still  undergo  profound  changes. 

1  Compt.  rend,  du  XIII6  Congres  internat.  de  Med.,  Paris,  1901.  Leube,  "  Ueber 
extrabuccale  Ernahrung,"  in  "  Deutsche  Klinik  am  Eingange  d.  XX.  Jahrhunderts," 
Wien  u.  Leipzig,  1901,  i,  S.  64. 

5—2 


68  Chapter  IV 

When  we  inject  milk,  blood  serum,  or  white  of  egg,  that  is  to  say, 
materials  very  rich  in  albuminoid  substances,  under  the  skin  or  into 
the  peritoneal  cavity  of  laboratory  animals,  we  find  that  after  a  time 
they  disappear.  At  the  same  time  they  give  rise  to  modifications  of 
the  organism  which  indicate  that  these  injected  substances  have  there 
undergone  profound  changes. 

After  injecting  eel's  serum  into  rabbits,  Th.  Tchistovitch1  found 
a  substance  in  the  blood  of  the  injected  animals  which  gave  a 
precipitate  with  eel's  serum.  Shortly  afterwards  Bordet58  observed 
that  the  blood  of  animals  into  which  he  had  injected  cow's  milk 
acquired  a  new  property :  it  gave  a  precipitate  with  this  milk,  a  con- 
dition never  observed  in  the  serum  of  untreated  animals. 

The  injection  of  white  of  egg  into  rabbits,  carried  out  by  Myers3 
and  Uhlenhuth4,  brought  about  the  same  changes  in  the  blood  serum. 
The  researches  of  the  latter  of  these  two  observers  have  for  our 
present  purpose  a  special  interest.  He  demonstrated  first  that  the  in- 
jection of  white  of  egg  into  the  peritoneal  cavity  of  rabbits  was  followed 
by  the  appearance  in  the  blood  serum  of  these  animals  of  a  substance 
which  precipitates  egg  albumen  in  vitro.  Uhlenhuth  then  obtained 
this  same  acquired  property  of  the  blood  in  rabbits  which  had  been 
made  to  swallow  a  considerable  quantity  of  the  white  of  hens'  eggs. 
Twenty-four  days  after  the  commencement  of  this  regimen  the  serum 
of  the  rabbits  precipitated  white  of  egg  in  the  test-tube.  This  example 
affords  a  marked  analogy  between  the  results  of  digestion  in  the 
alimentary  canal  and  those  of  resorption  into  the  tissues.  Uhlenhuth 
points  out,  indeed,  that  his  rabbits  which  received  the  injections  of 
white  of  egg  into  the  peritoneal  cavity  flourished  under  this  treatment. 

A  certain  number  of  similar  examples  are  now  recognised.  They 
all  indicate  that  various  nutritive  substances,  when  introduced  into 
the  peritoneal  cavity  or  under  the  skin  of  animals,  are  retained  there 
for  a  longer  or  shorter  time  and  are  subjected  to  certain  modifying 
influences  on  the  part  of  the  organism.  The  proof  that  these 
[74]  substances  are  not  eliminated  intact  by  the  kidneys  has  been 
furnished  by  a  large  number  of  experiments.  Recently  Lindemann5 
and  NeTedieff 6,  working  in  my  laboratory,  have  established  the  fact 

1  Ann.  de  Vlnst.  Pasteur,  Paris,  1899,  t.  xm,  p.  406. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1899,  t.  xin,  p.  225. 

3  CentralU.f.  Bakteriol  u.  Parasitenk.,  Jena,  1900,  Ite  Abt,  Bd.  xxvin,  8.  237. 

4  Deutsche  tried.  Wchmchr.,  Leipzig,  1900,  S.  734. 
6  Ann.  de  llnst.  Pasteur,  Paris,  1900,  t.  xiv,  p.  49. 
6  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  17. 


Resorption  of  the  formed  elements  69 

that  normal  blood  serum,  when  injected  under  the  skin  of  animals, 
does  not  provoke  albumiuuria  at  all,  or  at  least  produces  it  in  a  very 
insignificant  and  transitory  degree. 

The  mechanism  by  which  the  organism  modifies  these  nutritive 
substances,  introduced  by  a  channel  other  than  the  digestive  canal,  is 
not  as  yet  sufficiently  known;  and  is  therefore  not  easy  to  define. 
But  we  know,  very  definitely,  that  each  injection  of  serum,  whether 
of  white  of  egg,  milk  or  fatty  matter,  is  followed  by  a  rather  consider- 
able aseptic  inflammation  at  the  point  at  which  these  substances  are 
introduced.  We  might  conclude  from  this  that  the  organism  digests 
the  food  substances  outside  the  gastro-intestinal  canal,  by  means  of 
an  inflammatory  reaction.  In  order  to  determine  more  exactly  the 
phenomena  that  appear  under  these  conditions,  it  may  be  useful  to 
consider  first,  not  the  fluid  substances  but  the  solid  elements  that  are 
introduced  into  the  tissues  and  cavities. 

Let  us  begin  with  the  lower  animals  in  which  the  anatomical 
organisation  and  all  the  functions  are  of  a  much  more  simple 
character  than  they  are  in  the  Yertebrata.  In  my  Comparative 
Pathology  of  Inflammation  (Lecture  IV)  I  have  directed  some 
attention  to  the  digestion  of  the  Sponges. 

The  nutritive  substances — small  organisms — whether  they  may 
have  entered  by  the  small  openings,  so  numerous  on  the  surface  of 
Sponges,  or  have  been  introduced  through  a  rent  in  the  body  wall, 
undergo  the  same  fate.  They  are  seized  by  vibratile  or  amoeboid 
cells  which  ingest  the  food  and  digest  it  by  an  intracellular  digestion. 
These  two  kinds  of  cells,  which  come  under  the  category  of  Phagocytes, 
have  a  great  resemblance  to  one  another,  and  we  may  say  that 
digestion  and  resorption  are  two  very  closely  related  phenomena. 

When  we  examine  somewhat  higher  Invertebrata,  such  as  the 
Medusae  or  certain  other  Coelenterates,  we  can  still  trace  a  close 
analogy  between  the  true  digestion  of  the  food  that  goes  on  within 
the  epithelial  cells  of  the  entoderm  and  the  resorption  of  certain 
foreign  bodies  which  make  their  way  by  an  extra-buccal  channel  into 
the  intermediary  tissue.  Here  these  bodies  are  surrounded  by 
amoeboid  cells  which  fulfil  their  function  as  phagocytes  by  ingesting  [75] 
and  digesting  the  substances  that  have  come  from  outside. 

It  is,  here,  unnecessary  to  go  over  the  whole  gamut  of  the 
perfecting  of  the  organisation  of  the  Invertebrata,  in  its  relation 
to  the  resorption  of  foreign  bodies,  especially  as  it  has  already 
been  treated  in  my  Lectures  on  Inflammation.  Let  us  choose 


70  Chapter  IV 

merely  some  of  the  more  common  and  better-known  representatives 
of  the  Invertebrata  and  dwell  for  a  few  moments  on  the  phenomena 
manifested  in  their  organism,  into  the  midst  of  which  have  been 
introduced  a  few  nucleated  red  blood  corpuscles1. 

If  a  small  drop  of  defibrinated  blood  from  a  goose  be  injected 
beneath  the  skin  of  a  snail  and  another  under  the  skin  of  a  cockchafer 
larva,  the  red  corpuscles  are  disseminated  in  the  blood  fluid  which,  of 
itself,  is  incapable  of  modifying  them,  but  at  the  end  of  a  few  hours 
the  leucocytes  of  the  two  invertebrates  that  we  have  chosen  for  the 
experiment  will  have  ingested  a  certain  number  of  the  injected 
red  blood  corpuscles.  The  next  day  red  blood  corpuscles  are  still  to 
be  found  intact  in  the  blood  plasma,  but  the  great  majority  have  been 
devoured  by  the  leucocytes  (Fig.  13).  Inside  these  cells  the  red 
corpuscles  undergo  constant  and  marked  changes.  In  the  snail  they 
become  round  and  their  walls  permeable.  In  the  vacuoles  that  are 
produced  around  the  ingested  red  corpuscles  dissolved  haemoglobin 
is  found  (Fig.  14) ;  a  portion  of  this  colouring  matter  passes  into  the 
nucleus  of  the  red  corpuscles,  so  that  it  also  has  undergone  a  pro- 
found change  (Fig.  14).  Many  of  the  nuclei  become  emptied,  only 
the  peripheral  layer  remaining.  This  layer  and  the  membrane  of  the 
red  corpuscle  are  the  parts  that  resist  the  action  of  the  leucocytes 
longest  and  they  are  found  for  some  time  after  their  ingestion.  The 
white  corpuscles  of  the  snail,  having  devoured  one  or  more  red 
corpuscles,  may  themselves  become  the  prey  of  their  fellows. 

In  the  "  ver  blanc "  (French  popular  name  for  the  larva  of  the 

cockchafer)  the  phenomena  of  resorption  of  the  red  corpuscles  of  the 

goose  resemble  those  just  described.  The  blood  plasma  leaves  intact 

the  red  corpuscles  which  undergo  no  change  until  they  have  been 

ingested  by  the  leucocytes.      The  haemoglobin    diffuses    into   the 

leucocyte,  whilst  the  nucleus  and  the  membrane  persist  for  a  very 

[76]  considerable  period  (Fig.  15),  'though  they  lose  their  normal  aspect, 

shrivel,  and  become  transformed  into  an  irregular  mass  of  brown 

[77]  pigment  which  may  remain    in    the   substance    of  the    leucocyte 

(Fig.  I5,p)  for  weeks. 

Having  once  injected  goose's  blood  into  snails  and"vers  blancs," 
if  we  repeat  the  injection  several  times,  the  phenomena  observed  are 

1  The  resorption  of  the  red  blood  corpuscles  by  the  phagocytes  of  larvae  of 
starfish  (Bipinnarid)  and  of  Phyllirhoe  has  been  described  in  my  paper  on  intra- 
cellular  digestion  in  the  Invertebrates  in  Arb.  a.  d.  Zool.  Inst.  d.  Univ.  Wien,  1883, 
Bd.  v,  Hft  2,  8.  141. 


Resorption  of  the  formed  elements 


71 


invariably  the  same.  The  red  corpuscles  are  unacted  upon  by  the 
plasma  and  undergo  the  same  changes  within  the, leucocytes.  These 
changes  are  in  fact  comparable  to  those  described  in  the  preceding 


FIG.  13.  Leucocytes  of  a  cockchafer  larva 
containing  red  blood  corpuscles  of 
a  goose. 


FIG.  14.  Eed  blood  corpuscles  of  a  goose, 
free,  and  ingested  by  leucocytes  of  a 
snail  (Helix  pomatia),  24  hours  after 
their  injection. 


FIG.  15.  Leucocyte  of  a  cockchafer  larva, 
7  days  after  last  injection  of  goose's 
blood. 


FIG.  16.  Leucocyte  from  peritoneal  cavity 
of  a  gold-fish  after  ingesting  red  blood 
corpuscles  of  a  guinea-pig. 


chapter  in  discussing  the  intracellular  digestion  of  the  red  corpuscles 
by  the  intestinal  cells  of  the  Planarians.  In  both  cases  the  red  cor- 
puscles are  seized  by  amoeboid  cells  and  subjected  to  the  influence  of 


72  Chapter  IV 

their  contents.  In  the  intestinal  phagocytes  of  the  Planarian,  as  in 
the  phagocytes  of  the  blood  (leucocytes)  of  the  snail  and  "ver  blanc," 
the  haemoglobin  diffuses  through  the  wall  of  the  red  corpuscle, 
whose  most  resistant  parts  are  the  nucleus  and  the  membrane.  These 
resistant  residual  fragments,  impregnated  with  haemoglobin,  become 
brown  in  the  Planarian,  in  the  "ver  blanc,"  and  also,  but  in  a  less 
degree,  in  the  snail.  The  most  appreciable  difference  consists  in 
the  formation  of  excretory  vacuoles,  containing  concretions,  in  the 
Planarian,  and  the  absence  of  these  vacuoles  in  the  blood  phagocytes 
of  the  other  Invertebrata.  We  have,  however,  less  right  to  attribute 
a  fundamental  importance  to  this  difference,  in  that  the  phenomena  in 
the  Actinians,  which  ingest  the  red  blood  corpuscles  by  the  amoeboid 
cells  of  their  entoderm,  are  in  all  respects  (with  the  exception  of  the 
presence  of  these  special  excretory  vacuoles)  comparable  to  the 
phenomena  observed  in  the  Planarians.  From  the  fact  that  in  these 
two  examples  we  have  to  do  with  a  true  intracellular  digestion,  it 
must  be  admitted  that  the  modifications  of  the  red  blood  corpuscles 
within  the  phagocytes  of  the  blood  in  the  snail  and  in  the  larva  of  the 
cockchafer,  must  also  be  placed  in  the  same  category  of  phenomena. 

In  order  to  make  a  more  thorough  study  of  this  intracellular 
digestion  in  the  phagocytes  of  the  blood,  we  must  direct  our  attention 
to  larger  and  more  highly  organised  animals  than  the  snail  and  the 
"ver  blanc."  Let  us  take,  first,  an  example  among  the  inferior 
cold-blooded  Vertebrata.  The  red  blood  corpuscles  of  a  few  drops 
(0*25  c.c.)  of  the  blood  of  a  guinea-pig  injected  into  the  peritoneal  cavity 
of  a  gold-fish  (Cyprinus  auratus)  are  not  appreciably  changed  by  the 
peritoneal  fluid  itself ;  but  the  numerous  leucocytes  that  are  found  in 
[78]  the  peritoneal  fluid  seize  tfcim  and  ingest  them,  just  as  do  the  phago- 
cytes of  the  blood  of  Invertebrata,  or  the  intestinal  phagocytes  in  the 
Planarians  and  Actinians  in  the  case  of  the  red  blood  corpuscles  of 
the  goose.  Each  leucocyte  of  the  Cyprinus  ingests  several  red  blood 
corpuscles  and  subjects  them  to  intracellular  digestion.  The  stroina 
of  the  red  corpuscles  becomes  permeable ;  the  haemoglobin  diffuses 
into  the  nutritive  vacuoles  and  at  the  end  of  a  shorter  or  longer  period 
the  whole  is  dissolved  and  decolorised  (Fig.  16). '  Here  no  brown 
pigment  is  produced  and  the  red  corpuscles  are  completely  digested, 
leaving  no  "remains";  in  this  respect  differing  from  the  process  in  the 
Invertebrata  mentioned. 

This  result  depends,  probably,  partly  upon  the  more  feeble  resist- 
ance offered  by  the  non-nucleated  red  corpuscles  of  Mammals,  and 


Resorption  of  the  formed  elements  73 

partly  upon  the  more  active  digestive  power  of  the  leucocytes  of 
Fishes. 

As  the  result  of  several  injections  of  guinea-pig's  blood  into  the 
peritoneal  cavity  of  Cyprinus,  the  peritoneal  fluid  acquires  new 
properties1.  If,  a  fortnight  after  the  first  injection,  a  little  of  the 
peritoneal  exudation  in  the  gold-fish  be  withdrawn,  it  is  found  that 
a  drop  of  the  serum  which  floats  on  the  surface  produces,  almost 
immediately,  well-marked  agglutination  of  the  red  corpuscles  of  the 
guinea-pig,  this  being  soon  followed  by  the  rapid  solution  of  these 
red  blood  corpuscles  in  the  fluid.  This  new  property,  which  does  not 
exist  in  the  untreated  fish,  also  makes  its  appearance  in  the  blood 
serum  of  Cyprlni  treated  with  guinea-pig's  blood.  The  experiment 
is  very  successful  at  a  temperature  of  18° — 19°  C. 

As  the  solution  or  lysis  of  the  red  blood  corpuscles  in  the  serum 
is  exactly  like  that  which  takes  place  within  the  leucocytes  of 
Cyprinm,  we  are  justified  in  assuming  that,  in  both  cases,  it  is 
produced  by  the  same  substance.  And,  since  the  solvent  or  haemo- 
lytic  power  of  the  serum  is  only  acquired  as  the  result  of  the 
intracellular  digestion  of  the  red  blood  corpuscles  by  the  leucocytes, 
it  is  probable  that  the  solvent  substance  represents  the  intracellular 
ferment  derived  from  the  leucocytes. 

The  subject  we  have  just  broached  is  of  fundamental  importance 
in  connection  with  the  study  of  resorption  and  of  the  phenomena  of 
immunity  dependent  upon  it.  It  is  necessary,  therefore,  that  we 
should  go  more  fully  into  its  analysis.  With  this  object  we  must  first 
review  the  processes  that  go  on  during  resorption  in  the  higher 
animals  and  continue  our  examination  of  the  changes  that  injected  [79] 
or  extravasated  blood  undergoes  in  various  positions  of  the  organism. 

This  study  is  rendered  comparatively  easy  for  us  by  the  numerous 
researches  that  have  been  carried  out  by  pathological  anatomists  for 
the  purpose  of  ascertaining  the  fate  of  effusions  or  extravasations  of 
blood  so  frequently  met  with  in  disease.  It  has  long  been  known  that 
in  subcutaneous,  cerebral  and  other  haemorrhages,  or  in  hepatised 
lungs,  there  are  found  in  the  escaped  blood  a  great  number  of  cells 
containing  red  corpuscles.  As  was  mentioned  in  the  preceding  chapter, 
these  cells  were  evidently  amoeboid  cells  that  had  ingested  red  blood 
corpuscles.  To  Langhans2  especially  we  owe  a  detailed  study  of  the 

1  I  have  only  been  able  to  discover  the  haemolytic  property  of  the  serums  of 
Cyprinus  after  the  third  injection  of  guinea-pig's  blood. 

2  Virchow1*  Archie,  1870,  Bd.  XLIX,  S.  66. 


74  Chapter  IV 

phenomena  that  follow  extravasation  of  blood  produced  artificially  in 
the  subcutaneous  tissue  of  the  pigeon,  rabbit  and  guinea-pig.  In  all 
these  animals  the  haemorrhage  is  early  followed  by  exudative  inflam- 
mation, during  which  the  leucocytes  come  up  in  great  numbers  and 
ingest  the  red  blood  corpuscles  which  are  modified  in  the  interior  of 
the  leucocytes.  There  is  a  formation  or  deposition  of  pigment  and 
finally  all  traces  of  the  red  corpuscles  disappear.  In  Mammals  the 
pigment  is  brown  or  brownish,  just  as  it  is  in  the  Planarians  and 
in  the  "ver  blanc";  in  the  pigeon  it  is  green  and  resembles  that 
found  in  the  Actinians.  In  short  there  is  a  great  analogy  between 
the  resorption  of  red  corpuscles  and  the  true  intracellular  digestion  of 
the  red  blood  corpuscles  that  goes  on  in  the  intestinal  cells  of  the 
Invertebrata. 

But  what  is  the  nature  of  these  amoeboid  elements  that  intervene 
in  the  resorption  of  the  extravasated  blood  ?  At  the  period  when 
Langhans  carried  out  his  investigation,  we  were  unable  to  differentiate 
the  cells  at  all  satisfactorily.  It  is  only  since  the  publication  of 
Ehrlich's  classic  researches  on  the  white  corpuscles  that  we  have 
been  able  to  bring  more  order  into  this  question.  Thanks  to  the  use 
of  various  aniline  stains,  Ehrlich  was  able  to  arrange  the  leucocytes 
found  in  the  Vertebrata  into  several  definite  groups. 

The  question  has  already  been  touched  upon  in  our  eighth  lecture 
on  inflammation ;  it  is  therefore  unnecessary  to  treat  it  here  at  length. 
We  must,  however,  before  entering  on  the  analysis  of  the  essential 
phenomena  in  the  resorption  of  cells,  as  we  now  understand  them, 
give  a  rapid  survey  of  the  different  varieties  of  amoeboid  cells  that  are 
found  in  the  Vertebrata. 

[80]  Beside  mobile  amoeboid  cells,  represented  by  several  forms  of 
white  corpuscles,  we  must  distinguish  fixed  amoeboid  cells.  These 
are  permanently  fixed  in  certain  situations  in  the  body;  this,  however, 
in  no  way  prevents  them  from  throwing  out  amoeboid  processes  in 
various  directions  and  seizing  foreign  bodies  or  certain  elements 
of  the  same  organism.  The  nerve  cells,  the  large  cells  of  the  splenic 
pulp  and  of  the  lymphatic  glands,  certain  endothelial  cells,  the  cells 
of  the  neuroglia,  and  perhaps  some  connective  tissue  cells,  belong  to 
the  category  of  fixed  amoeboid  cells.  All  these  elements,  under 
certain  conditions,  are  able  to  ingest  solid  bodies ;  consequently,  they 
act  as  phagocytes.  With  the  exception  of  the  cells  of  the  nerve 
centres,  all  these  fixed  phagocytes  are  of  mesoblastic  origin.  It  has 
been  much  discussed  whether  certain  processes  of  the  nerve  cells  may 


Resorption  of  the  formed  elements  75 

not  really  serve  to  seize  foreign  bodies  and  carry  them  into  the  cell 
contents.  It  appears  to  us  that  sometimes  they  undoubtedly  do  fulfil 
this  function.  For  example,  it  is  only  by  means  of  such  amoeboid 
movements  that  leprosy  bacilli  can  be  introduced  into  the  interior  of 
ganglion  cells  and  cells  of  the  spinal  cord1.  We  must  not  dwell 
on  this  question,  as  the  phagocytic  property  of  the  nerve  elements 
plays  no  part  in  the  resorption  of  cells.  On  the  other  hand,  the 
neuroglia  cells  contribute  largely  to  this  process  and  their  phagocytic 
function  is  now  admitted  by  many  observers2. 

For  long  the  large  "  dust "  cells  of  the  respiratory  channels  were 
looked  upon  as  being  epithelial  cells  which  were  capable  of  ingesting 
carbon  particles,  micro-organisms  and  other  foreign  bodies.  The  re- 
searches of  N.  Tchistovitch,  carried  out  in  my  laboratory  more  than 
twelve  years  ago,  made  it  evident  that  these  elements  are  nothing 
more  than  white  corpuscles  that  have  immigrated  into  the  alveoli 
and  bronchi. 

It  is  probable  that  the  same  is  the  case  as  regards  the  stellate  cells 
of  the  liver,  known  as  Kupffer's  cells.  First  described  by  Kupfler 
as  cells  of  a  nervous  type,  having  long  processes,  they  were  later 
recognised  by  several  observers  as  belonging  to  the  endothelial  [81] 
tissue  of  the  blood  vessels  of  the  liver.  Kupffer3  himself  has  ac- 
cepted this  view  and  in  his  recently  published  monograph  on  these 
stellate  cells,  he  describes  them  as  endothelial  cells  that  have 
retained  their  independence.  Some  researches  on  the  resorption 
of  blood,  of  which  I  shall  speak  shortly,  have  led  me  to  think 
that  these  cells  are  nothing  but  white  corpuscles  that  have  been 
arrested  in  the  hepatic  capillaries.  I  have  asked  Mesnil,  head  of 
my  laboratory,  to  study  this  question  for  me.  His  investigation  is  not 
yet  concluded,  but  the  demonstration  already  made  that  the  livers 
of  guinea-pig  embryos  and  new-born  rabbits  do  not  possess  any 
KupiFer's  cells  is  an  argument  in  favour  of  my  hypothesis. 

Certain  white  corpuscles  have  undoubtedly  been  often  mistaken 
for  epithelial  or  connective  tissue  cells.  We  must  not  conclude  from 
this,  however,  that  these  elements  are  never  capable  of  sending  out 
amoeboid  processes  and  of  ingesting  foreign  bodies.  It  would,  how- 
ever, be  useful  to  collect  new  and  incontestable  proofs  of  the 

1  Soudake witch,  Ziegler's  Beitr.  z.  path.  Anat.,  Jena,  1888,  Bd.  n,  S.  129,  and 
Babes,  "  Untersuchungen  iiber  den  Leprabacillus,"  Berlin,  1898,  S.  58. 

2  Marinesco,  Compt.  rend.  Soc.  de  Jliol.,  Paris,  1896,  p.  726. 

3  Arch.f.  mikr.  Anat.,  Bonn,  1899,  Bd.  LIV,  S.  254. 


76  Chapter  IV 

accuracy  of  this  thesis.  In  spite  of  this  uncertainty,  it  may  be 
accepted  as  fully  demonstrated,  that  certain  fixed  amoeboid  cells, 
such  as  the  large  elements  of  the  splenic  pulp,  of  the  lymphatic 
glands,  and  of  the  omentum,  play  an  important  part  in  the  resorption 
of  cells.  It  is  there  that  elements  filled  with  red  corpuscles  and 
white  corpuscles  in  process  of  being  destroyed  are  so  often  found. 
Just  as  certain  fixed  cells  do  not  function  as  true  phagocytes, 
so  also  in  some  leucocytes  this  function  is  undoubtedly  absent.  The 
suggestion  has  been  made  several  times  that  any  cell  element, 
provided  it  be  young,  is  capable  of  ingesting  foreign  bodies.  The 
examination  of  white  corpuscles  proves  exactly  the  contrary.  The 
smaller  white  corpuscles  found  in  fairly  large  numbers  in  the  blood 
and  the  lymph,  and  which  are  commonly  known  as  lymphocytes  or 
small  lymphocytes,  are  simply  leucocytes  with  very  little  protoplasm 
which  in  this  state  never  fulfil  phagocytic  functions.  It  is  only  when  it 
becomes  older,  when  its  nucleus,  single  and  rich  in  chromatin,  becomes 
surrounded  by  an  ample  layer  of  protoplasm,  that  the  lymphocyte 
becomes  capable  of  ingesting  and  resorbing  foreign  bodies.  Several 
[82]  authors,  with  Ehrlich  at  their  head,  still  assign  to  these  larger  cells 
the  same  name— lymphocytes.  Others,  however,  give  them  the  name 
of  large  mononuclear  cells.  Confusion  is  thus  possible,  especially  as 
Ehrlich  includes  under  the  same  term  the  large  mononucleated  leuco- 
cyte, a  very  rare  form  of  cell  in  human  blood,  which  is  distinguished 
by  the  greater  staining  capacity  of  its  nucleus.  To  avoid  this  incon- 
venience I  propose  to  designate  the  large  lymphocytes  by  the  name 
of  blood  maCrophages  and  lymph  macrophages  (haemo  macrophages, 
lymphomacrophages).  This  term  is  preferable  to  that  of  mononuclear 
leucocytes,  especially  as  in  exudations  we  frequently  meet  with  macro- 
phages with  two  and  even  several  sharply  separated  nuclei.  Giant  cells, 
moreover,  are  nothing  but  polynucleated  macrophages.  On  the  other 
hand,  the  leucocytes  so  often  designated  by  the  name  of  poly  nuclear  in 
reality  contain  but  a  single  nucleus.  Even  Ehrlich,  who  introduced  this 
term,  acknowledged  its  imperfection  but  he  retained  it  for  some  time 
because  it  was  already  very  extensively  used  and  could,  he  thought, 
give  rise  to  no  misunderstanding.  In  his  excellent  work  on  anaemia, 
published  jointly  with  Lazarus1,  he  now  agrees  that  the  name  of  "cells 
with  polymorphous  nuclei "  would  be  more  exact. 

1  Ehrlich  u.  Lazarus,  "Die  Anaemie,"  in  Nothnagel's  "Specielle  Pathologic  u. 
Therapie,"  Wien,  1898,  Bd.  vm,  I48*  Theil,  S.  49.  Cf.  the  authorised  English  trans- 
lation, "Histology  of  the  Blood,"  Cambridge,  1900,  p.  74. 


Resolution  of  tlie  formed  elements  77 

These  polynaorpho-nuclear  leucocytes  are  very  numerous  in  the 
blood  and  in  many  exudations  and  are  distinguished  by  the  greater 
selective  affinity  of  their  nucleus  for  basic  aniline  dyes  and  by 
a  certain  tendency  of  the  protoplasm  to  become  stained  by  acid 
aniline  colours,  such  as  eosin.  The  true  macrophages  are  without 
granulations,  but  the  "  polymorpho-nuclear "  leucocytes  contain 
many.  These  granulations  are  sometimes  "  eosinophile,"  "pseudo- 
eosinophile"  (or  "  amphophile ")  or  even  "  neutrophile  "  (as  in  man 
and  the  horse). 

These  two  main  groups  of  leucocytes  are  generally  distributed  in 
the  Vertebrate, ;  and  we  already  meet  with  them  in  one  of  the  lowest 
vertebrate  forms — the  Ammocoetes  (the  larva  of  the  lamprey).  The 
macrophages  of  this  fish  present  all  the  principal  characters  of  the  %~ 
group  to  which  they  belong  (protoplasm  without  granules,  easily 
stained  with  methylene  blue,  large  nucleus  rich  in  nuclear  juice).  In  [S3] 
the  "polynuclear"  forms  in  this  lower  vertebrate  the  protoplasm  does 
not  stain  with  methylene  blue,  but  assumes  a  faint  rosy  tint  with 
eosin ;  the  single  nucleus  is  divided  into  several  lobes.  In  Vertebrates 
which  are  much  higher  in  the  scale  these  characters  change.  Thus  in 
the  cayman  (Alligator  mississipiemis),  according  to  the  researches  of 
Madame  Podwyssotsky,  carried  out  in  my  laboratory,  the  two  great 
varieties  of  leucocytes  are  readily  found  in  the  blood,  lymph  and  exu- 
dations. The  macrophages,  however,  especially  in  the  exudations,  are 
very  often  furnished  with  two  or  several  nuclei,  whilst  the  small  leuco- 
cytes possess  only  a  single  nucleus,  which  is  not  divided  into  lobes.  In 
spite  of  this  peculiarity  the  two  groups  are  readily  distinguished.  The 
staining  reactions  of  the  macrophages  are  identical  with  those  of  the 
corresponding  corpuscles  in  all  the  other  Vertebrata ;  whilst  the  small 
leucocytes,  in  spite  of  the  absence  of  a  polymorphous  nucleus,  are  easily 
recognised  by  their  eosiuophile  granulations  and  by  the  special  affinity 
of  the  nucleus  for  basic  aniline  dyes.  Under  these  circumstances  it 
would  be  quite  inappropriate  to  designate  those  leucocytes,  which  are 
really  polynuclear,  that  is  to  say,  possessing  two  or  several  nuclei,  by 
the  name  of  " mononuclear,"  and  to  reserve  the  name  of  "poly- 
nuclear "  for  the  small  corpuscles  which  possess  only  a  single  nucleus 
undivided  into  lobes.  For  this  reason  it  is  much  more  rational  to 
retain  for  these  so-called  polynuclear  cells  my  proposed  name  of 
microphages.  Moreover,  the  microphages  are  true  phagocytes.  It 
was  formerly  thought  that  the  eosinophile  leucocytes,  such  as  the 
"  *  overfed '  cells  (Mastzellen) "  of  Ehrlich,  which  are  identical  with 


78  Chapter  IV 

the  clasmatocytes  of  Ranvier,  never  ingested  foreign  bodies.  But, 
(especially  after  the  researches  of  Mesnil1),  we  have  been  compelled 
to  change  our  opinion  on  this  point.  The  true  eosinophile  cells  are 
able  to  devour  foreign  bodies,  especially  micro-organisms,  and  must 
therefore  be  regarded  as  phagocytes  belonging  to  the  group  of 
microphages. 

It  is  the  peculiar  merit  of  Ehrlich  and  of  his  school  that  they  have 
thoroughly  established  the  fact  that,  in  Mammals  at  any  rate,  the  two 
principal  groups  of  white  cells  are  distinguished,  amongst  other 
characters,  by  the  diversity  of  their  origin.  The  lymphocytes  and  the 
mononuclear  cells  are  developed  in  the  spleen  and  lymphatic  glands, 
whilst  the  "  polynuclear  "  cells  arise  from  the  granular  mononucleated 
myelocytes  of  the  bone  marrow.  This  is  now  generally  accepted  as 
[84]  applicable  in  the  great  majority  of  cases.  In  Ammocoetes,  however, 
the  two  chief  varieties  of  leucocytes  arise  from  one  and  the  same 
organ,  regarded  by  several  observers  as  a  kind  of  primitive  spleen, 
which  runs  along  and  in  part  surrounds  the  intestine.  Mesnil  has 
been  good  enough  to  make  sections  of  this  primitive  organ  in  which 
it  may  be  demonstrated  that  the  macrophages  and  the  microphages 
in  the  larva  of  the  lamprey  have  the  same  seat  of  origin.  Frog 
tadpoles  and  Cartilaginous  Fishes  also  possess  microphages  which  do 
not  arise  from  the  bone  marrow,  since  in  them  this  tissue  is  completely 
absent.  But  even  in  Mammals,  at  least  in  certain  pathological  con- 
ditions, Dominici2,  in  a  research  executed  with  much  care  and  a 
perfect  technique,  has  demonstrated  the  myelogenous  transformation 
going  on  in  the  spleen.  Thus  in  the  adult  rabbit  affected  with 
septicaemia  by  the  typhoid  bacillus,  he  found  in  the  spleen  de- 
velopmental centres  of  amoeboid  elements  which,  normally,  appear 
to  develop  in  the  bone  marrow  only,  i.e.  the  megacaryocytes,  or  large 
cells  with  budding  nuclei,  the  neutrophile  myelocytes  (amphophiles), 
basophiles  and  eosinophiles. 

The  mesoblastic  phagocytes  of  the  Vertebrata  are  divided,  then, 
into  fixed  phagocytes — the  macrophages  of  the  spleen,  endothelia, 
connective  tissue,  neuroglia,  and  muscle  fibres — and  free  phagocytes. 
These  latter  are  sometimes  haemo-  or  lympho-macrophages,  sometimes 
microphages.  The  fixed  macrophages  and  the  free  macrophages  re- 
semble one  another  so  greatly  that  it  is  very  often  extremely  difficult, 
if  not  impossible,  to  differentiate  them.  For  this  reason  it  is  often 

1  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  301. 

2  Arch,  de  med.  exper.,  Paris,  1901,  t.  xm,  p.  1. 


Resorption  of  the  formed  elements  79 

very  useful,   when  the  exact  origin  of  a  large  phagocyte  is  not 
known,  simply  to  name  it  "  macrophage." 

The  two  principal  groups  of  phagocytes — (1)  fixed  and  free 
macrophages,  (2)  microphages — are  distinguished  not  only  by  their 
morphological  characters  ;  they  also  give  evidence  of  very  marked 
physiological  differences.  All  phagocytes  are  endowed  with  amoeboid 
movement  which  allows  them  either  to  move  about  freely  or  merely 
to  put  out  protoplasmic  processes.  These  movements  are  regulated  by 
a  very  great  sensitiveness,  often  different  in  the  two  groups.  Besides 
a  tactile  sense,  the  phagocytes  possess  a  kind  of  sense  of  taste  or 
chemiotaxis  which  enables  them  to  distinguish  the  chemical  com- 
position of  the  substances  with  which  they  come  in  contact.  The  [85] 
existence  of  this  chemiotaxis  could  be  anticipated  from  the  moment 
that  an  important  part  in  the  life  of  the  organism  began  to  be 
ascribed  to  the  amoeboid  cells.  Leber1,  Massart  and  Charles  Bordet2 
have,  however,  demonstrated  it  by  rigorous  experiment.  Following 
the  method  used  by  Pfeffer  to  demonstrate  the  chemiotaxis  of  the 
vegetable  spermatozoids  and  of  Bacteria,  these  investigators  intro- 
duced into  the  bodies  of  higher  (rabbits  and  guinea-pigs)  and  lower 
(frogs)  Vertebrates  small  glass  tubes  filled  with  different  solutions 
(peptone,  broth,  salts,  bacterial  products,  etc.).  The  leucocytes, 
guided  by  their  positive  chemiotaxis,  made  their  way  into  the 
tubes  and  there  formed  plugs  which  were  often  very  voluminous; 
when,  on  the  other  hand,  the  chemical  composition  of  the  solutions 
excited  their  negative  chemiotaxis,  the  leucocytes  avoided  the  tubes. 

Having  acquired  information  as  to  the  chief  characters  of  the 
leucocytes,  we  may  ask,  To  which  group  do  those  amoeboid  cells, 
which,  according  to  the  observations  of  Langhans  and  many  other 
investigators,  bring  about  the  resorption  of  the  red  corpuscles  of  the 
blood,  belong?  This  resorption  goes  on  more  rapidly  and  is  observed 
much  better  if,  instead  of  introducing  blood  of  the  same  species  into 
any  part,  we  inject  defibrinated  blood,  or  red  blood  corpuscles  from 
which  the  serum  has  been  removed  by  washing,  from  another  species 
of  Vertebrate.  It  will  be  found  best  to  inject  the  nucleated  red 
corpuscles  of  lower  Vertebrates  into  Mammals,  or  (as  already  de- 
scribed above)  to  introduce  the  non-nucleated  red  blood  corpuscles 
of  Mammals  into  lower  Vertebrates.  In  all  these  cases  the  injection 

1  Fortschr.    d.    Med.,    Berlin,    1888,  Bd.  vi,  &  460;    "Die    Entstehung    der 
Entziindung,"  Leipzig,  1891. 

2  Journ.  publ.  par  la  Soc.  roy.  d.  Sc.  med.  et  nat.  de  Bruxelles,  1890,  3  Feb. 


80  Chapter  IV 

of  such  blood  or  corpuscles  sets  up  an  aseptic  inflammation  which 
attracts  a  large  number  of  free  phagocytes  to  the  seat  of  injection. 
In  subcutaneous,  peritoneal  or  intraocular  exudations  produced 
under  these  conditions,  we  find,  in  addition  to  a  number  of  micro- 
phages,  many  macrophages.  Whilst  the  former  ingest  the  injected 
red  corpuscles  merely  in  isolated  cases,  the  positive  chemiotaxis 
of  the  macrophages  manifests  itself  much  more  actively.  In  the 
resorption  of  the  red  blood  corpuscles  the  more  important  part  is 
played  by  the  macrophage.  To  get  a  clear  idea  of  the  phenomena 
[86]  that  accompany  this  resorption,  let  us  take  a  concrete  example. 
Inject  defibrinated  goose's  blood  into  the  peritoneal  cavity  of  guinea- 
pigs1.  During  the  first  few  hours  after  injection  the  oval  nucleated 
red  corpuscles  are  found  intact  in  the  fluid  of  the  peritoneal  lymph. 
The  plasma,  by  itself,  exercises  no  destructive  or  solvent  action  on 
the  red  corpuscles  of  the  goose. 

Immediately  after  the  injection  the  lymph  of  the  peritoneal  cavity 
begins  to  show  important  changes.  The  white  corpuscles  which,  in 
the  normal  condition,  are  fairly  abundant,  disappear  almost  com- 
pletely ;  some  small  lymphocytes  presenting  their  ordinary  aspect  may 
indeed  be  found,  but  the  few  macrophages  and  the  microphages 
that  remain  show  signs  of  very  grave  lesions.  They  lose  their 
mobility,  run  together  into  clumps  and  become  incapable  of  ingesting 
foreign  bodies.  At  this  moment  the  phagocytes  undergo  a  critical 
change  which  we  have  designated  by  the  name  of  phagolysis.  This 
condition  lasts  for  about  an  hour,  sometimes  it  continues  longer, 
according  to  case  and  circumstance,  but  after  this  the  peritoneal 
fluid  becomes  filled  with  leucocytes  that  have  newly  come  on  to  the 
scene.  These  cells  make  their  way,  by  diapedesis,  through  the 
walls  of  the  congested  vessels  of  the  peritoneum.  A  true  aseptic 
inflammation  is  produced  which  induces  an  exudation  of  a  large 
number  of  white  corpuscles,  amongst  which  are  found  microphages 
and  still  more  numerous  macrophages.  The  latter  show  a  very 
pronounced  positive  chemiotaxis  towards  the  injected  red  corpuscles 
of  the  goose.  Soon  after  their  appearance,  that  is  to  say  two  or  three 
hours  after  the  injection  of  the  blood,  the  macrophages  send  out  very 
small  protoplasmic  processes  and  aflix  them  to  the  surface  of  the  red 
corpuscles.  There  follows  an  aggregation  of  the  macrophages  of  the 
guinea-pig  with  the  red  corpuscles  of  the  goose  and  characteristic 
•  masses,  in  which  can  be  recognised  both  kinds  of  cells,  are  produced. 
1  Ann.  de  VInst.  Pasteur,  Paris,  1899,  t.  xm,  p.  742. 


Resorption  of  the  formed  elements  81 

This  union  with  the  very  small  pseudopodia  is  the  first  stage  in  the 
ingestion  of  the  red  corpuscles  by  the  macrophages  (Fig.  17).  The 
red  corpuscle,  seized  by  amoeboid  processes,  passes  into  the  interior 
of  the  macrophage.  This  macrophage  seldom  rests  contented  with 
ingesting  a  single  red  corpuscle.  Usually  it  devours  a  large  number 
and  sometimes  enormous  macrophages  may  be  seen  filled  with  a  score 
of  red  corpuscles. 

If  the  quantity  of  goose's  blood  injected  into  a  guinea-pig  is  large 
(5 — 7  c.c.),  the  ingestion  of  red  corpuscles  by  the  macrophages  con-  [87] 
tinues  for  a  considerable  period — often  for  three  to  four  days. 
During  the  whole  of  this  time  a  certain  number  of  the  red  corpuscles 
remain  free  in  the  peritoneal  plasma,  but,  in  spite  of  this  prolonged 
stay,  none  of  them  undergo  extracellular  solution. 


FIG.   17.    Macrophage  of  guinea-pig  in        FIG.  18.    Macrophage  of  guinea-pig  in 
process  of  devouring  and  digesting  red  the  act  of  ingesting  and  digesting  red 

blood  corpuscles  of  goose.  corpuscles  of  goose.  Infra  vitam  stain- 

ing with  neutral  red. 

The  red  blood  corpuscles,  anchored  by  the  amoeboid  processes  of 
the  macrophages,  at  first  present  a  normal  appearance.  Later  their 
membrane  begins  to  wrinkle,  but  as  soon  as  they  have  passed  within 
the  phagocytes  the  wrinkles  disappear  and  the  corpuscles  regain  their 
normal  aspect.  If  a  little  neutral  red  solution  be  added  to  a  drop  of  peri- 
toneal exudation  (Fig.  18)  we  observe  that  the  nucleus  of  the  ingested 
red  corpuscle  and  even  its  contents  are  stained  red,  whilst  the  red  cor- 
puscles adherent  to  the  surface  of  the  phagocytes  retain  their  normal 
yellow  colour.  This  reaction  enables  us  to  see  that  the  red  corpuscles 
are  seized  by  the  macrophages  whilst  still  in  their  normal  condition, 
but  that  they  undergo  a  change  immediately  after  they  have  been 
B.  6 


82  Chapter  IV 

ingested.  Little  by  little  the  devoured  corpuscles  are  digested  within 
the  phagocytes.  The  haemoglobin  diffuses  into  the  contents  of  the 
macrophage  through  the  stroma,  which  has  become  permeable ;  the 
nucleus  of  the  ingested  red  corpuscle  also  becomes  stained  by 
the  haemoglobin.  Part  of  this  colouring  matter  is  excreted  by  the 
[88]  phagocyte.  The  body  of  the  red  corpuscle  is  pretty  soon  digested, 
but  the  nucleus,  impregnated  with  haemoglobin,  persists  for  a  much 
longer  period.  It  divides  into  several  fragments,  recognisable  by  their 
yellow  colour,  and  in  certain  cases  these  remnants  of  red  corpuscles 
may  be  met  with  for  weeks  in  the  interior  of  the  macrophages.  These 
macrophages  do  not  remain  permanently  in  the  peritoneal  fluid. 
Some  (3 — 4)  days  after  injection  the  lymph  of  the  peritoneum 
contains  only  leucocytes  that  have  newly  come  up  and  which 
contain  neither  red  corpuscles  nor  their  remains.  We  must  open 
the  guinea-pig  to  find  any  macrophages  that  have  devoured  red 
corpuscles.  They  are  to  be  met  with  in  large  numbers  in  the 
glandular  portion  of  the  omentum,  in  the  mesenteric  glands,  in  the 
liver  and  in  the  spleen.  They  are  fairly  easily  recognised  by  the 
characteristic  aspect  of  the  debris  of  the  red  blood  corpuscles. 
Having  devoured  the  red  corpuscles  the  macrophages  leave  the 
peritoneal  fluid  and  the  digestion  is  completed  in  the  positions 
just  mentioned.  In  the  liver  they  are  seen  as  large  mononuclear 
cells  often  with  highly  developed  processes.  In  this  condition  they 
remind  one  of  Kupffer's  stellate  cells — a  fact  that  suggested  to  me 
the  idea  that  these  elements  are  nothing  but  white  corpuscles  which 
have  immigrated  into  the  vessels  of  the  liver. 

Following  up  the  fate  of  the  macrophages  that  have  resorbed 
the  red  blood  corpuscles,  we  find  them  in  the  large  hepatic  vessels, 
in  the  vena  cava  and  even  in  the  blood  of  the  heart.  But  in 
these  latter  situations  they  contain  merely  a  few  scarcely  recognisable 
traces  of  their  prey.  These  phagocytes,  which  left  the  blood  during 
the  inflammation  that  followed  the  injection  of  red  corpuscles  of 
the  goose,  re-enter  it,  having  fulfilled  their  function,  during  the  final 
period  of  the  resorption.  This  resorption  must  undoubtedly  be 
regarded  as  an  intracellular  digestion.  When  we  compare  the 
essential  phenomena  taking  place  inside  the  macrophages  containing 
red  blood  corpuscles  with  those  we  have  described  in  the  intestinal 
phagocytes  of  the  Planarians  or  Actinians  after  a  meal,  the  analogy 
between  the  two  becomes  very  apparent.  In  both  cases  the  red  blood 
corpuscles  undergo  a  marked  change  which  results  in  a  diffusion  of 


Resolution  of  the  formed  elements  83 

the  haemoglobin.  The  membrane  and  nucleus  of  the  red  blood 
corpuscles  persist  longer  but  they  also  are  ultimately  digested.  The 
excretion  of  haemoglobin  from  the  phagocytes,  just  mentioned  in  the 
case  of  the  macrophages  of  the  guinea-pig,  is  also  observed  in  the 
Actinians,  whose  coelenteric  cavity  is  tinted  by  a  rose-coloured  solution. 

We  have  seen  that  in  the  Actinians  intracellular  digestion  takes  [89] 
place  in  a  distinctly  acid  medium,  whilst  in  the  intestinal  cells  of  the 
Planarians  it  takes  place  in  one  that  is  only  weakly  acid.  The 
macrophages  of  the  guinea-pig,  during  the  resorption  of  red  blood 
corpuscles  of  the  goose,  carry  on  the  digestive  process  in  a  medium 
which  shows  a  still  weaker  acidity.  When  made  to  ingest  granules  of 
blue  litmus  there  is  no  change  of  colour.  Nor  does  alizarin  sulpho- 
acid  give  any  reaction,  probably  owing  to  the  fact  that  it  exerts  a 
toxic  action  on  the  protoplasm  of  the  macrophages.  If,  however,  we 
add  to  a  drop  of  the  peritoneal  exudation  of  a  guinea-pig,  containing 
macrophages  filled  with  red  blood  corpuscles  of  the  goose,  a  little  of 
Ehrlich's  1  %  solution  of  neutral  red,  the  red  brick  tint  at  once  makes 
its  appearance  in  the  content  of  these  phagocytes.  This  coloration 
is  identical  with  that  described  in  the  Amoebae  which  digest  Bacteria 
or  in  the  intestinal  phagocytes  of  the  Planarians.  It  may,  then,  be 
regarded  as  an  indication  of  weak  acidity.  This  coloration  is  main- 
tained for  some  hours,  after  which  it  gives  place  to  complete  decolora- 
tion, a  phenomenon  that  must  be  attributed,  as  in  many  other  cases, 
to  the  neutralisation  of  the  acid  by  the  alkaline  protoplasm  that  has 
been  macerated  in  the  fluid  after  the  death  of  the  macrophages. 

The  example  we  have  chosen — the  destruction  of  red  blood 
corpuscles  of  the  goose  by  the  macrophages  of  the  guinea-pig- 
may  serve  as  a  prototype  of  the  resorption  of  formed  elements  in 
general.  If,  instead  of  red  blood  corpuscles  of  the  goose,  we  inject 
into  the  guinea-pig's  peritoneal  cavity  pigeon's  or  fowl's  blood,  the 
essential  phenomena  will  be  the  same.  The  red  blood  corpuscles  will 
always  induce  positive  chemiotaxis,  especially  of  the  macrophages, 
which  in  turn  will  ingest  the  nucleated  red  corpuscles.  It  may  be 
that  in  certain  cases,  when  fowl's  blood  containing  red  corpuscles 
that  are  not  very  resistant  is  injected,  a  certain  number  of  the  cor- 
puscles immediately  undergo  a  partial  solution  in  the  peritoneal  fluid1. 

1  Krtmpecher  (Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  lte  Abt.,  Jena,  1900, 
Bd.  xxvin,  S.  588)  has  obtained  a  serum  which  was  even  capable  of  altering  the 
nuclei  of  the  red  corpuscles  of  the  frog.  These  nuclei  must  be  much  less  resistant 
than  those  of  the  red  blood  corpuscles  of  birds,  such  as  the  goose,  fowl  and  pigeon. 

6—2 


84  Chapter  IV 

Here  also  the  stromas  and  the  nuclei  of  all  the  red  blood  corpuscles, 
as  well  as  many  of  the  corpuscles  unacted  upon  by  the  plasma  of 
the  phagolysed  exudation,  undergo  digestion  inside  the  macrophages. 
[90]  When,  instead  of  blood,  we  inject  white  corpuscles  from  the  bone 
marrow,  spleen  or  lymphatic  glands  of  animals  into  the  peritoneal 
cavity,  we  may  still  observe  their  final  disappearance  in  the  macro- 
phages. The  spermatozoa  of  man  or  of  various  mammals  (bull,  rabbit, 
guinea-pig,  etc.),  when  injected  into  the  peritoneal  cavity  of  the 
guinea-pig  or  rabbit,  are  well  adapted  for  this  line  of  investigation. 
Here  again  the  immediate  result  of  injection  is  the  very  marked 
phagolysis  of  the  leucocytes.  This  phenomenon  gives  place  to  an 
exudative  inflammation  which  brings  into  the  peritoneal  cavity 
a  number  of  phagocytes.  These,  especially  the  macrophages  and  in 
a  much  smaller  degree  the  microphages,  devour  the  spermatozoa 
which  in  no  case  are  dissolved,  even  partially,  in  the  plasma  of  the 
exudation.  The  macrophage  seizes  the  spermatozoa  which  sometimes, 
by  the  active  movements  of  their  flagella,  exhibit  great  vitality.  At 
the  end  of  several  hours  all  the  spermatozoa  are  found  inside  phago- 
cytes where  they  are  completely  destroyed.  The  flagellum  is  digested 
first,  but  the  head  and  medial  portion  soon  suffer  the  same  fate. 
Neutral  red  reveals  the  feebly  acid  reaction,  perhaps  with  even  more 
distinctness  than  in  the  case  of  the  red  blood  corpuscles. 

The  rtfsumtf  of  Langhans'  investigation  given  in  this  chapter  would 
lead  us  to  expect  that  resorption  in  the  subcutaneous  tissue  will 
follow  the  same  rules  as  that  going  on  in  the  peritoneal  cavity.  As 
a  matter  of  fact,  blood  injected  at  this  position  sets  up  a  diapedesis 
of  phagocytes  which  ingest  the  red  blood  corpuscles.  In  some  cases 
only  is  there  a  partial  solution  of  these  corpuscles  in  the  fluid  of 
the  subcutaneous  exudation.  It  is  for  this  reason  that  goose's  blood, 
injected  under  the  skin  of  a  guinea-pig,  gives  rise  to  a  fluid  exudation 
coloured  a  bright  rose  red  by  the  dissolved  haemoglobin.  This 
haemoglobin  is  derived  from  red  blood  corpuscles  which  are  damaged 
by  the  goose's  blood  serum  that  was  added  to  the  plasma  of  the 
exudation.  The  stroma  and  nuclei  of  the  red  blood  corpuscles  cannot, 
however,  be  dissolved  in  this  fluid.  They  undergo  the  same  fate  as 
the  red  corpuscles  that  have  remained  intact,  that  is  to  say  they  are 
ingested  by  the  macrophages  which  immigrate  into  the  subcutaneous 
tissue  and  which  finally  digest  all  these  elements.  The  cells,  less  fragile 
than  certain  red  corpuscles,  are,  in  the  subcutaneous  tissue,  as  in  the 
peritoneal  cavity,  destroyed  solely  in  the  interior  of  the  phagocytes. 


Resorption  of  the  formed  elements  85 

The  analogy  between  the  modifications  undergone  by  the  red 
blood  corpuscles  and  other  cells  inside  the  macrophages  and  the 
changes  that  take  place  in  the  intestinal  cells  of  Planarians  and 
Actinians,  suggests  that  the  resorption  of  formed  elements  must  [91] 
undoubtedly  be  regarded  as  a  true  intracellular  digestion.  It  would, 
however,  be  a  very  important  matter  to  be  able  to  support  this  con- 
clusion by  even  more  convincing  proofs.  The  study  of  the  artificial 
digestion  that  is  observed  in  vitro  in  the  case  of  the  macerated  mesen- 
terial  filaments  of  Actinians  has  furnished  a  very  valuable  argument  in 
favour  of  the  enzymatic  nature  of  intracellular  digestion.  Animal 
exudations  are  not  well  adapted  for  this  special  line  of  study.  We  can 
only  obtain  them  as  the  result  of  the  injection  of  different  substances, 
solid  or  fluid,  which  are  greedily  absorbed  by  phagocytes.  If  we  collect 
the  exudations  at  a  moment  when  the  number  of  these  cells  is  still 
considerable  we  must  withdraw  along  with  them  many  digestive  sub- 
stances which  interfere  with  our  observation.  We  may  therefore  with 
advantage  turn  our  attention  to  masses  of  phagocytes  collected  in 
organs.  As  it  is  mainly  the  macrophages  which  effect  the  resorption 
of  cells,  it  is  evident  that  we  must  choose  the  centres  where  they  are 
formed  in  order  to  investigate  the  digestive  ferments.  Let  us  take, 
then,  the  lymphatic  glands  of  the  mesentery,  the  glandular  portion  of 
the  omentum  and  the  spleen,  the  three  pre-eminently  macrophagic 
organs,  and  let  us  see  if,  with  an  extract  of  them,  prepared  with 
physiological  salt  solution  (075  %  of  sodium  chloride),  any  digestive 
effect  is  to  be  obtained. 

Macerate  the  three  organs  mentioned  of  a  guinea-pig  and  mix 
the  extracts  thus  obtained  with  red  blood  corpuscles  of  the  goose, 
corpuscles  that  have  already  given  us  information  in  connection  with 
the  phenomena  of  resorption  in  the  living  organism.  In  almost  all 
the  guinea-pigs  a  solution  of  the  red  blood  corpuscles  of  the  goose 
by  the  extract  of  the  glandular  portion  of  the  omentum  may  be 
observed.  The  mesenteric  glands  likewise  give  an  extract  which 
in  most  cases  has  a  solvent  action.  The  extract  from  the  spleen  is 
only  active  in  a  limited  number  of  cases.  In  all  these  examples  the 
extracts  from  macrophagic  organs  bring  about  the  solution  of  the 
haemoglobin,  but  leave  intact  the  membrane  and  nucleus  of  the 
corpuscles.  In  this  respect  there  exists,  then,  a  certain  difference 
between  this  and  the  digestion  of  red  corpuscles  in  the  macrophages 
of  exudations,  where  the  membrane  and  even  the  nucleus  are  in  the 
end  completely  dissolved.  This  difference  may  be  explained  by  the 


86  Chapter  IV 

fact  that  in  the  preparation  of  the  extract  in  physiological  salt  solution, 
one  part  only  of  the  soluble  digestive  ferment  may  be  set  at  liberty. 

The  solvent  action  of  extracts  of  macrophagic  organs  must  in  fact 
[92]  be  attributed  to  the  presence  of  a  soluble  ferment  in  the  cells  of 
which  these  organs  are  made  up.  As  the  diastases  are  distinguished, 
in  general,  by  their  great  sensitiveness  to  heat,  we  tried  the  action  of 
our  extracts  after  a  preliminary  heating,  when  it  was  found  that  a 
temperature  of  56°  C.,  applied  for  three  quarters  of  an  hour,  com- 
pletely abolished  the  solvent  action  of  the  extracts  upon  the  red  blood 
corpuscles  of  the  goose.  The  soluble  ferment  of  macrophagic  organs, 
to  which  we  propose  to  give  the  name  of  macrocytase1  or  macrophage 
ferment,  is  in  many  respects  analogous  to  the  actino-diastase  of 
Mesiiil,  described  in  the  preceding  chapter. 

With  a  view  to  obtain  more  complete  information  on  the  cytases 
I  suggested  to  Tarassewitch  that  he  should  make  a  detailed  study 
of  them ;  this  he  has  carried  out  in  my  laboratory.  He  has  demon- 
strated that  the  macrophagic  organs  of  other  mammals  than  the 
guinea-pig,  especially  those  of  the  rabbit  and  dog,  exert  the  same 
solvent  action  on  the  red  blood  corpuscles.  He  has  also  established 
the  fact  that  this  action  applies  not  only  to  the  red  corpuscles  of  the 
goose  but  extends  also  to  those  of  several  other  birds  and  mammals. 
Tarassewitch  succeeded  in  confirming  the  injurious  action  of  heat  on 
macrocytase.  Extracts  of  macrophagic  organs  which  contain  much 
debris  in  suspension,  when  heated  for  an  hour  at  55°'5C.  in  certain 
cases  lose  their  solvent  property  for  red  blood  corpuscles  ;  sometimes 
this  temperature  brings  about  merely  a  weakening  of  the  macrocytase. 
In  order  to  destroy  it  surely  and  completely,  the  suspensions  must  be 
heated  at  58°'5 — 62°  C.  for  an  hour.  If,  however,  instead  of  heating 
the  entire  suspension,  we  first  pass  it  through  filter  paper,  the  clear 
fluid  filtrate  is  deprived  of  its  diastatic  action  even  after  it  has  been 
heated  at  55°'5C.  for  three  quarters  of  an  hour. 

Of  all  the  other  organs  of  which  extracts  have  been  kept  in  pro- 
longed contact  with  the  red  blood  corpuscles  of  birds,  the  pancreas 
alone  lias  shown  a  very  well-marked  digestive  action.  Extracts  of  the 

1  Some  years  ago  it  was  proposed  to  give  the  name  of  cytase  to  the  ferments 
which  digest  cellulose.  Thus  Laurent,  in  the  work  analysed  in  the  second  chapter, 
applies  it  to  the  ferment  secreted  by  the  bacilli  which  attack  the  vegetable  membrane. 
We  think  that  the  cellulose  ferment  should  be  designated  by  the  name  of  cellulosase 
and  that  the  name  of  cytase  would  be  more  suitable  for  a  soluble  ferment  which 
digests  the  cells. 


Resorption  of  the  formed  elements  87 

salivary  glands  exerted  a  feeble  solvent  action  on  a  certain  quantity  of 
the  red  corpuscles.     The   other  organs,  such  as  the  liver,  kidneys,  [93] 
brain,  spinal  cord,  ovary,  testicles,  suprarenal  capsules  and  placenta, 
exercised  no  such  action.    Even  bone  marrow,  in  agreement  with  my 
results  published  some  years  ago,  showed  itself  quite  inactive. 

The  blood  serum  of  guinea-pigs  which  I  employed  in  my  researches, 
as  well  as  that  of  the  animals  examined  by  Tarassewitch,  has  not 
shown  itself  capable  of  dissolving  the  red  blood  corpuscles  of  the 
goose,  although  the  macrophagic  organs  dissolve  them  easily.  It  has 
long  been  known,  however,  that  the  serum  of  the  blood  of  many 
animals  will  destroy  the  red  corpuscles  of  a  different  species.  This 
demonstration  was  afforded  during  the  period  when  attempts  were 
being  made  to  transfuse  the  defibrinated  blood  of  mammals,  espe- 
cially of  the  sheep,  into  man.  This  practice  had  to  be  abandoned, 
in  consequence  of  the  difficulties  resulting  from  the  solution  of  the 
human  red  corpuscles.  Later,  Daremberg1  and  Buchner2  set  them- 
selves to  study  this  haemolytic  action  of  serums  systematically.  They 
found  that  it  was  due  to  a  particular  substance  to  which  Buchner 
gave  the  name  of  alexine  or  protective  substance.  Of  indeterminate 
chemical  composition,  this  substance  is  allied  to  albuminoid  sub- 
stances. It  is  destroyed  when  heated  to  55° — 56°  C.  and  only  acts  in 
the  presence  of  certain  salts.  When  these  salts  are  removed  from  the 
serum  by  dialysis,  it  loses  its  haemolytic  power;  but  as  soon  as  the 
salts  are  replaced  in  proper  proportion  this  power  reappears.  Later, 
Buchner3  compared  the  action  of  alexine  to  that  of  soluble  ferments 
and  referred  it  to  the  category  of  the  digestive  diastases.  According 
to  him  the  same  alexine  is  capable  of  dissolving  the  red  blood 
corpuscles  of  several  species  of  Vertebrates.  Bordet4,  in  a  series  of 
researches  made  in  the  Pasteur  Institute,  confirmed  this  view.  He 
came  to  the  conclusion  that  the  alexines  of  the  various  species  of 
animals  differ  from  one  another.  Thus,  the  alexine  of  the  blood 
serum  of  the  rabbit  is  not  the  same  as  that  found  in  the  serum 
of  the  guinea-pig  or  dog.  Nevertheless  each  of  these  alexines  is 
capable  of  exerting  a  solvent  action  on  the  red  blood  corpuscles  of 
several  species. 


1  Arch,  de  med.  exper.,  Paris,  1891,  t.  in,  p.  720. 

2  Verhandl.  d.  X.  Congr.f.  inn.  Med.,  Wiesbaden,  1892. 

3  Munchen.  med.  Wchmchr.,  1900,  S.  1193. 

4  Ann.  de  Hnst.  Pasteur,  Paris,  1899,  t.  xm,  p.  273 ;  1901,  t.  xv,  p.  312. 


88  Chapter  IV 

[94]  Ehrlich  and  Morgenroth1,  in  a  series  of  memoirs  on  the  solution  of 
red  blood  corpuscles,  have  combated  the  idea  that  there  is  only  a  single 
alexine  in  one  and  the  same  serum.  Moreover,  they  state  that  alexine 
always  requires  for  its  action  the  aid  of  another  substance,  and  that 
matters  are  much  more  complicated  than  at  first  sight  appears.  They 
maintain  that  in  each  normal  serum  a  number  of  different  substances 
are  found,  each  one  of  which  only  attacks  a  single  species  of  red  blood 
corpuscle.  They  point  out  that  the  solution  of  the  red  corpuscles  by 
the  normal  serum  takes  place  through  the  combined  action  of  two 
different  substances  and  cite  several  cases  where  a  normal  serum,  after 
being  heated  to  55°  C.  and  so  deprived  of  its  haemolytic  power,  again 
becomes  capable  of  dissolving  the  red  corpuscles  when  some  normal 
serum  from  another  species,  which  of  itself  is  destitute  of  the  solvent 
property,  is  added  to  it.  Let  us  quote  an  example  from  Ehrlich 
and  Morgenroth.  The  normal  serum  of  the  goat  readily  dissolves 
the  red  blood  corpuscles  of  the  rabbit  and  guinea-pig,  but  if  heated 
for  half  an  hour  at  55°  C.,  it  loses  this  power.  On  the  other  hand, 
the  normal  serum  of  many  horses  shows  itself  powerless  to  dissolve 
the  red  corpuscles  of  these  rodents.  Here,  then,  are  two  serums, 
equally  incapable  of  effecting  the  solution  of  the  red  corpuscles  of  the 
rabbit  and  guinea-pig.  Yet,  when  they  are  mixed  together  and  to 
them  a  few  drops  of  blood  from  one  of  the  rodents  cited  is  added, 
haemolysis  takes  place  readily.  The  heated  goat's  serum  then,  has, 
retained  in  it  something  that  resists  a  temperature  of  55°C.,  a  sub- 
stance which,  by  itself,  leaves  the  red  blood  corpuscles  intact :  but 
which,  when  combined  with  a  second  substance  present  in  the  horse's 
serum,  causes  their  solution.  Ehrlich  gives  to  the  first  substance, 
that  is  to  say  that  found  in  the  heated  goat's  serum,  the  name  of 
intermediary  body  ("  Zwischenkorper  ").  The  second  substance,  pre- 
sent in  the  unheated  horse's  serum,  is  designated  by  him  the  comple- 
ment. In  order  that  a  normal  serum  may  dissolve  the  red  corpuscles, 
it  is  not  sufficient  that  it  should  possess  a  single  substance,  the  alexine 
of  Buchner.  It  must,  to  exert  this  action,  contain  two  distinct  sub- 
stances which  are  very  often  found  together  in  the  same  normal  serum. 
Unheated  goat's  serum  was  only  capable  of  dissolving  the  red  blood 
corpuscles  of  the  rabbit  because  a  particular  complement  and 
intermediary  substance  were  both  present.  Deprived  of  its  comple- 
ment at  55°  C.,  the  serum  is  solvent  only  when  we  add  to  it  another 

[95]  substance  that  is  contained  in  the  normal  serum  of  a  different  species 
1  Berl  kiln.  Wchnschr.,  1899,  SS.  6  and  481. 


Resorption  of  the  formed  elements  89 

(horse).  Continuing  their  researches  in  this  direction,  Ehrlich  and 
Morgenroth  have  come  to  the  conclusion  that  the  normal  serum  of  a 
single  species  may  contain  several  intermediary  substances,  each  one 
acting  on  a  single  species  of  red  blood  corpuscles.  Further,  that 
normal  serum  must  contain  several  or  even  many  different  comple- 
ments. 

Ehrlich  and  Morgenroth  carried  on  researches  on  the  intermediary 
substances  in  normal  serums  and  found  several  in  addition  to  that 
already  mentioned.  The  serum  of  the  normal  dog  readily  dissolves 
the  red  blood  corpuscles  of  the  guinea-pig.  When  heated  to  57° C.  it 
loses  this  property;  but  with  the  addition  of  normal  guinea-pig's  serum 
the  property  is  regained.  In  the  serum  of  the  normal  dog  there  exists, 
then,  besides  the  complement,  at  least  one  intermediary  substance. 
The  same  result  can  be  obtained  with  several  combinations  of  serums 
of  normal  mammals,  heated  or  unaltered1.  Yet  it  often  happens,  as 
Ehrlich  and  Morgenroth  themselves  point  out,  that  the  demonstration 
of  the  presence  of  the  intermediary  substance  in  normal  serums  is 
accompanied  with  marked  difficulties.  Bordet,  also,  who  has  studied 
this  question  very  thoroughly,  often  failed  completely  in  his  attempts 
to  make  normal  serums,  that  were  incapable  of  producing  haemolysis, 
active  by  the  addition  of  heated  serums  of  other  species  of  animals. 
Thus  he  observed  that  normal  fowl's  serum  readily  dissolves  the  red 
corpuscles  of  the  rabbit  When  heated  to  55° — 56° C.  this  serum 
loses  its  haemolytic  power  which  cannot  be  restored  by  the  addition 
of  any  normal  serum.  He  thinks  therefore  that,  in  this  example, 
haemolysis  is  produced  solely  by  the  alexine,  without  the  assistance 
of  any  intermediary  substance  in  the  serum  of  the  normal  fowl. 
P.  Miiller2,  whilst  confirming  Bordet's  experimental  results,  considers 
that,  in  this  case  also,  there  is  the  intervention  of  an  intermediary 
substance.  When  he  mixed  heated  fowl's  serum  with  a  small  quantity 
of  unaltered  fowl's  serum  the  solution  of  the  red  corpuscles  of  the  [96] 
rabbit  is  not  brought  about.  When,  however,  instead  of  adding  a 
little  imheated  normal  fowl's  serum,  he  added  the  same  quantity  of 

1  Ehrlich  and  Morgenroth,  "  Ueber  Haemolysine,"  II,  Berl.  klin.  Wchnschr.,  1899, 
S.  481.     The  following  are  the  combinations  found  by  these  observers  :  heated  calf's 
serum  with  normal  serum  dissolves  the  red   blood  corpuscles  of  the  guinea-pig; 
heated  rabbit's  serum  plus  sheep's  serum  dissolves  the  red  blood  corpuscles  of  the 
sheep ;   heated  serum  of  rabbit  with  the  addition  of  goat's   serum   dissolves  the 
red  corpuscles  of  the  goat ;  heated  sheep's  serum  with  guinea-pig's  serum  produces 
haemolysis  of  the  red  corpuscles  of  the  guinea-pig. 

2  CentralU.f.  Bakteriol  u.  Parasitenk.,  ite  Abt,  Jena,  1901,  Bd.  xxix,  S.  175. 


90  Chapter  IV 

serum  from  a  fowl  previously  treated  with  physiological  salt  solution, 
the  red  corpuscles  of  the  rabbit  were  dissolved  without  any  diffi- 
culty. Miiller  explains  this  difference  as  due  to  the  fact  that  the 
serum  of  the  treated  fowl  contains  more  complementary  substance 
than  does  that  of  the  normal  fowl. 

We  see,  then,  from  this  example  that  the  analysis  of  the  pheno- 
mena taking  place  in  the  solution  of  the  red  corpuscles  by  normal 
serums  is  beset  with  very  great  difficulties.  For  this  reason  it  is 
much  more  profitable  to  make  researches  in  this  direction,  using  more 
active  serums,  where  the  demonstration  of  the  two  substances  can 
be  made  simply  and  exactly.  This  desideratum  has  been  supplied  by 
J.  Bordet,  when  preparateur  in  our  laboratory ;  he  described  an  easy 
method  of  increasing  the  haemolytic  power  of  serums. 

As  stated  above,  guinea-pigs  that  have  received  an  intraperitoneal 
injection  of  goose's  blood  digest  the  corpuscles,  although  the  peri- 
toneal fluid  exerts  no  haemolytic  action.  In  vitro,  the  extract  of  their 
macrophagic  organs  certainly  dissolves  the  red  corpuscles,  whilst  the 
blood  serum  usually  fails  to  do  so.  Now,  if  a  second  or  a  third 
injection  of  goose's  blood  be  made  into  the  peritoneal  cavities  of  the 
same  guinea-pigs,  partial  solution  of  the  corpuscles  takes  place  in  the 
peritoneal  plasma  and  the  serum  of  the  blood  acquires  new  properties  : 
it  becomes  capable  of  clumping  the  red  corpuscles,  that  is  to  say  of 
agglutinating  them  ;  afterwards  it  dissolves  them  in  vitro. 

J.  Bordet1  has  shown  that  the  injection  of  the  blood  of  one  species 
of  Vertebrate  (mammal  or  bird)  into  the  peritoneal  cavity  or  under  the 
skin  of  an  animal  of  a  different  species,  always  produces  in  the  blood 
serum  of  the  latter  the  haemolysing  substance.  This  haemolysing 
substance  is  specific  or  nearly  so,  that  is  to  say  it  dissolves  the  red 
corpuscles  of  the  species  which  has  furnished  the  injected  blood  and 
also,  but  more  feebly,  the  red  corpuscles  of  allied  species.  Conse- 
quently, with  guinea-pig's  serum,  treated  with  goose's  blood,  we 
obtain  the  greatest  solvent  action  on  the  red  corpuscles  of  the  goose, 
though  there  is  a  certain  haemolysis  of  the  red  corpuscles  of  some 
other  birds.  This  rule,  thoroughly  established  by  Bordet,  has  been 
the  starting-point  for  a  large  number  of  researches  on  haemolysis 
[97]  and  amongst  others  of  those  which  bear  on  the  intermediary  sub- 
stance of  normal  bloods. 

Bordet  demonstrated  very  definitely  a  fact  of  fundamental  import- 
ance— that  in  the  blood  serums  of  animals  treated  with  blood  from  a 
1  Ann.  de  VInst.  Pasteur,  Paris,  1898,  t.  xn,  p.  688. 


Resorption  of  the  formed  elements  91 

different  species,  there  exist  two  distinct  substances  which  only  dis- 
solve the  red  blood  corpuscles  when  they  are  combined.  Here  the 
duality  of  the  haeraolytic  agent  cannot  be  doubted,  as  it  may  in 
certain  examples  of  normal  serums.  Each  time  that  we  deprive  the 
serum  of  a  treated  animal  of  its  solvent  action  by  heating  at  55° — 
56°  C.,  this  property  can  be  restored  to  it  with  certainty  by  the  addi- 
tion of  a  little  normal  serum  which,  by  itself,  is  incapable  of  bringing 
about  haemolysis.  The  heated  serum  of  these  injected  animals  loses 
the  power  of  dissolving  the  corresponding  red  corpuscles,  but  it  re- 
tains its  other  acquired  property — the  agglutination  of  the  corpuscles. 
The  red  corpuscles,  aggregated  into  voluminous  masses  quite  visible 
to  the  naked  eye,  remain  intact  indefinitely,  if  left  in  the  prepared 
and  heated  serum.  But  as  soon  as  we  add  to  them  a  trace  of  normal 
blood  (taken  from  one  of  a  number  of  species  of  Vertebrates),  the 
solution  of  the  red  corpuscles  is  not  long  in  taking  place.  Under 
these  conditions  an  action  of  two  substances  is  set  up ;  one  of  these 
substances  is  found  in  the  heated  serum  of  the  injected  animal,  and 
the  other  in  unheated  normal  serum.  The  first  of  these  substances 
which  not  only  resists  a  temperature  of  55° — 56°  C.,  but  stands,  with- 
out alteration,  heating  to  60° — 65°  C.,  corresponds  to  the  intermediary 
substance  of  Ehiiich.  By  Bordet  it  has  been  termed  "substance 
sensibilisatrice1."  The  second  substance,  a  common  one,  found  in 
normal  serums  and  destroyed  at  55° — 56°  C.,  is  the  alexine  of  Buchner 
and  of  Bordet,  or  the  complement  of  Ehrlich. 

The  ease  with  which  one  can  demonstrate  the  co-operation  of  two 
substances  in  the  haemolysis  by  the  serums  of  animals  treated  with 
the  blood  of  a  different  species,  is  due  to  the  fact,  that  during  the 
course  of  this  treatment  the  animal  organism  produces  a  quantity 
of  an  intermediary  or  sensibilising  substance.  In  fresh  animals 
which  have  not  been  treated,  it  is  often  very  difficult  to  demonstrate 
the  presence  of  this  substance.  Bordet  has  established  the  fact  that  [98] 
the  serum  of  animals  which  have  been  injected  several  times  with  the 
blood  of  a  different  species,  contains  almost  the  same  amount  of 
alexine  as  does  untreated  serum.  On  the  other  hand,  the  sensibilising 
substance  makes  its  appearance  in  large  quantity  as  the  result  of 
these  injections.  Von  Dungern2  has  confirmed  this  result  and  con- 

1  Among  the  synonyms  of  this  substance,  resistant  to  the  action  of  heat,  we  may 
mention  the  following :  haemolytic  antibody,  preventive  substance,  immunising  body 
(Tmmnnkorper  of  Ehrlich),  amboceptor  (Ehrlich),  philocytase  (Metchnikoff ),  desmon 
(London),  copula  (P.  Miiller). 

2  Miinchen.  med.  Wchnschr.,  1900,  S.  677. 


92  Chapter  IV 

tributes  the  interesting  fact  that  the  sensibilising  substance  is  found 
even  in  great  excess  in  the  serum  of  treated  animals.  When  he  adds 
to  this  serum  blood  that  has  not  been  heated,  he  produces  a  haemo- 
lysis that  is  more  than  thirty  times  as  active  as  when  the  serum  of 
the  prepared  animal  alone  is  used.  From  the  quantitative  point  of 
view,  then,  there  is  no  relation  between  the  amount  of  the  two  sub- 
stances in  the  serum  of  prepared  animals. 

It  may  be  suggested  that  the  sensibilising  or  intermediary  sub- 
stance is  the  same  as  that  which  produces  the  agglutination  of  the 
red  corpuscles.  But  careful  researches  have  thoroughly  demonstrated 
the  difference  between  the  two  substances  that  have  this  character  in 
common,  both  resist  heating  to  55° — 60°  C.  and  even  beyond  this  point. 

Having  established  this  co-operation  of  two  substances  in  haemo- 
lysis the  intimate  mechanism  of  their  action  was  next  studied.  Here 
I  must  give  pride  of  place  to  the  discovery  by  Ehrlich  and  Morgenroth 
that  the  intermediary  (or  sensibilising)  substance  links  itself  to  its 
corresponding  red  corpuscles.  A  serum,  capable  of  dissolving  the 
red  corpuscles  of  a  different  species,  is  heated  to  56°  C.  which  causes 
it  to  lose  this  solvent  property.  When  a  certain  number  of  these 
corpuscles  are  added  to  it,  such  corpuscles  remain  intact  although 
they  are  agglutinated.  It  is  sufficient,  after  some  hours  of  contact,  to 
centrifugalise  the  mixture  in  order  to  separate  a  limpid  serum  from 
the  mass  of  red  corpuscles,  the  former  being  now  entirely  deprived  of 
its  intermediary  substance,  that  is  to  say  it  has  become  incapable  of 
dissolving  the  red  corpuscles  even  with  the  addition  of  a  large  quantity 
of  the  "  complement "  (normal  serum,  unheated).  On  the  other  hand, 
the  red  corpuscles,  having  fixed  (linked)  all  the  intermediary  sub- 
stance, dissolve  very  rapidly  when  placed  in  contact  with  normal 
serum  which  contains  the  necessary  quantity  of  the  complement  (or 
alexine).  This  fundamental  experiment  has  been  confirmed  and 
repeated  by  many  observers  and  has  now  become  classic.  The  idea 
that  the  intermediary  (or  sensibilising)  substance  links  itself  to  the 
red  corpuscle,  without  dissolving  it,  is  generally  accepted  and  may  be 
regarded  as  permanently  settled.  We  should  do  well,  then,  instead 
[99]  of  designating  by  all  sorts  of  synonyms  the  substance 'in  serums  which 
resists  the  action  of  a  temperature  of  55° — 65°  C.,  to  apply  to  it,  once 
for  all,  the  name  of  fixative  substance  or  simply  that  of  fixative. 
This  name  is  short,  expresses  the  essential  character  of  the  substance 
and  gives  rise  to  no  misunderstanding,  as  do  the  other  names  proposed 
up  to  the  present  (amongst  them  that  of  philocytase  employed  by 
myself  in  some  of  my  earlier  publications). 


Resorption  of  the  formed  elements  93 

Another  of  Ehrlich  and  Morgenroth's  experiments  has  furnished 
the  proof  that  the  complement  unaided  does  not  fix  itself  to  the  red 
corpuscles.  A  normal  serum,  unheated,  which,  by  itself,  is  quite  as 
incapable  of  dissolving  the  red  corpuscles  as  the  fixative  alone,  is 
mixed  with  some  defibrinated  blood.  After  the  centrifugalisation  of 
this  mixture,  it  is  easy  to  demonstrate  that  the  supernatant  fluid 
has  lost  none  of  its  complement  (alexine),  whilst  the  red  corpuscles 
have  fixed  none. 

If,  instead  of  an  inactive  serum,  we  take  a  serum  which  is  capable 
of  dissolving  the  red  corpuscles  and  which  consequently  contains  the 
two  haemolysing  substances,  and  if  we  place  it  in  contact  with  the 
corresponding  red  corpuscles,  at  a  temperature  between  0°  and  3C  C., 
the  solution  will  not  take  place  (Ehrlich  and  Morgenroth).  Under 
these  conditions  the  fixative  certainly  attaches  itself  to  the  red 
corpuscles,  but  the  alexine  remains  in  solution,  unused.  It  is  only 
necessary,  however,  to  heat  the  mixture  up  to  30°  C.  to  bring  about 
rapid  haemolysis. 

From  their  very  ingenious  experiments,  as  a  whole,  Ehrlich  and 
Morgenroth  conclude  that  the  fixative  possesses  two  different  affini- 
ties :  one  for  the  red  corpuscle  and  another  for 
the  complement.     Of  these  two  affinities  the 
stronger  is  that  which  links  it  to  the  red  cor- 
puscle, for  this  is  manifested  at  a  very  low 
temperature.     In  order  that  the  fixative  may 
combine  with  the  complement  a  much  higher 
temperature  is  requisite.    Ehrlich  comes  to  the 
conclusion  that  the  molecule  of  the  fixative 
possesses  two  haptophore  groups,   or  groups         Flo  19     schema  of 
capable  of  chemical  combination.    The  first  of          Ehriich's  theory. 
these  links  it  to  a  corresponding  molecule  of  the      c,  complement  (alexine, 
red  corpuscle  to  which  he  gives  the  name  of         cytase)— am,    ambo- 
receptor ;  the  second  combines  the  fixative  with         cep*or  (fixa*ive)-f're- 

,.        •  ,        „  ..  .  i  .     ,i  .  ceptor  of  the  red  cor- 

the  molecule  of  the  complement  and  in  this  way         puscie. 

introduces   the  latter  into  the  red  corpuscle.      (After  Levaditi  in  the 

These  investigators  give  a  diagram  which  greatly         Presse  mtdicaie.) 

facilitates  the  understanding  of  their  hypothesis 

(Fig.  19).    They  seek  to  prove  that  the  combinations  of  the  fixative 

with  the  red  blood  corpuscle  and  with  the  complement  follow  the  law 

of  definite  multiples  and  that  these  phenomena  must,  in  consequence,  [100] 

be  looked  upon  as  being  of  a  purely  chemical  character. 


94  Chapter  IV 

The  hypothesis  advanced  by  J.  Bordet  does  not  accord  very  well 
with  the  theory  we  have  just  set  forth.  He  could  never  convince 
himself  that  the  fixative  combines  with  the  complement.  He  was  of 
opinion  rather  that  the  fixative,  retained  by  the  corpuscle,  exercises 
upon  it  a  mordant  action  which  enables  it  to  absorb  the  alexine.  The 
alexine  is  supposed  to  attach  itself  to  the  sensibilised  red  blood  cor- 
puscle as  a  dye  attaches  itself  to  a  mordanted  element.  Bordet  rests 
his  interpretation  mainly  on  the  fact  that  the  absorption  of  alexine 
by  the  sensibilised  corpuscles  does  not  follow  the  elementary  laws  of 
chemical  combination,  especially  those  of  definite  multiples. 

Nolf1  has  sought  to  define  more  accurately  the  part  played  by 
these  two  substances  in  the  solution  of  the  red  blood  corpuscles. 
He  agrees  with  Bordet,  that  in  this  phenomenon  the  fixative  plays 
the  same  part  that  the  mordants  do  in  dyeing.  Linked  to  the  red 
corpuscle  the  fixative  renders  it  more  greedy  for  alexine,  exactly  as 
the  mordant  facilitates  the  fixation  of  the  dye  on  the  fibre  of  the 
textile  fabric.  Under  these  conditions  the  alexine  (complement), 
finding  itself  in  large  quantity  inside  the  red  corpuscle,  exercises 
upon  it  its  hydrating  action,  thus  bringing  about  the  diffusion  of 
the  haemoglobin  and  often  even  the  solution  of  the  corpuscular 
stroma. 

Nolf  compares  the  solvent  action  of  alexine  upon  the  red  corpuscle 
to  that  of  certain  mineral  salts,  such  as  ammonium  chloride.  He 
passes  in  review  the  various  properties  of  alexines  and  finds  them 
very  similar  to  the  solvent  action  of  certain  salts.  Even  the  pecu- 
liarity of  alexine,  of  remaining  inactive  at  a  temperature  of  0° — 3°C., 
is  shared  by  ammonium  chloride  which,  alone  of  all  the  salts  studied 
by  Nolf,  exercises  no  solvent  action  under  these  conditions.  But  Nolf 
found  it  impossible  to  push  these  analogies  further,  and  especially  to 
sensibilise,  by  the  fixative,  the  red  corpuscles  to  the  action  of  quan- 
tities, which  were  of  themselves  inactive,  either  of  ammonium  chloride 
or  of  any  other  salt. 

[101]  London'2  hoped  by  fresh  experiments  to  solve  the  problem  of  the 
mode  of  action  of  the  two  substances  which  act  in  haemolysis.  He 
pronounced  in  favour  of  the  theory  that  they  entered  into  chemical 
combination  with  the  red  corpuscles.  But  the  facts  accumulated 
up  to  the  present  do  not  enable  us  to  make  a  positive  statement 
as  to  the  exact  nature  of  the  reaction  which  is  set  up  during  the 

1  Ann.  de  FInst.  Pasteur,  Paris,  1900,  t.  xiv,  p.  656. 

2  Arch.  d.  sc.  biol.  (russes),  1901,  t.  vm,  pp.  281  and  323. 


Resorption  of  the  formed  elements  95 

solution  of  the  red  blood  corpuscles ;  this  is  not  astonishing  in  view  of 
the  fact  that  it  is  impossible  to  isolate  the  haemolysing  substances  in 
a  pure  state. 

It  may,  however,  be  admitted  that  the  action  of  alexine  (comple- 
ment) comes  under  the  category  of  phenomena  that  are  produced  by 
soluble  ferments.  Buchner1  maintains  that  there  is  an  analogy  be- 
tween this  substance  and  the  diastases  (or  enzymes) ;  Bordet2,  from 
the  appearance  of  his  first  publications  on  haemolysis,  has  expressed 
himself  in  favour  of  this  view.  Ehrlich  and  Morgenroth3,  in  their 
two  first  memoirs,  very  distinctly  put  forward  the  same  idea.  "We 
shall  not  deceive  ourselves" — they  say — "if  we  attribute  to  the 
addiment  (syn.  complement,  or  alexine)  the  character  of  a  digestive 
ferment."  In  one  of  their  last  memoirs4  they  no  longer  express  them- 
selves in  so  decided  a  fashion.  Nevertheless  we  are  still  quite  justified 
in  maintaining  this  proposition.  The  substance  which  dissolves  the 
red  blood  corpuscles  of  Mammals  or  a  portion  only  of  those  of  Birds, 
undoubtedly  presents  very  great  analogies  to  the  digestive  ferments. 
As  has  been  mentioned  repeatedly,  it  is  very  sensitive  to  the  action 
of  heat  and  is  completely  destroyed  by  heating  for  one  hour  at  55° C. 
In  this  respect  it  closely  resembles  the  macrocytase  of  macrophagic 
organs  which  also  dissolves  red  corpuscles.  As  it  is  the  macrophages 
which  ingest  and  digest  the  red  blood  corpuscles  in  the  organism,  it 
is  evident  that  alexine  is  nothing  but  the  macrocytase  which  has 
escaped  from  the  phagocytes  during  the  preparation  of  the  serums. 

We  know  that  the  leucocytes  contain  quite  a  series  of  soluble 
ferments  of  which  some  are  set  at  liberty  after  the  blood  has  been 
drawn  from  the  vessels.  It  is  thus  that  plasmase,  or  fibrin-ferment, 
is  set  free  from  the  leucocytes  to  combine  with  fibrinogen  to  produce  [102] 
the  clot.  This  is  not  the  only  soluble  ferment  of  leucocytic  origin. 
It  has  been  known  for  some  time  that  in  addition  to  this  coagulating 
ferment  the  leucocytes  contain  ferments  which  are  especially  digestive 
or  decoagulating.  Thus  Rossbach5  has  demonstrated  the  presence  of 
amylase  in  the  leucocytes  of  different  organs,  especially  the  tonsils. 
ArthiiS'has  confirmed  this  discovery  and  Zabolotny6  has  completed  it 
by  his  ^observations  on  the  phenomena  which  appear  in  the  peritoneal 

1  Miinchen.  med.  Wchnschr.,  1900,  S.  1193. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1898,  t.  xn,  p.  688  ;  1899,  t.  xm,  p.  273. 

3  Berl  klin.  Wchnschr.,  1899,  SS.  6  and  481. 

4  Berl  klin.   Wchnschr.,  1900,  8.  682. 

6  Deutsche  med.  Wchnschr.,  Leipzig,  1890,  S.  389. 

6  Arch,  russes  d.palh.,  etc.,  St.-Petersb.,  1900,  t.  iv,  p.  402. 


96  Chapter  IV 

cavity  of  animals  into  which  wheat  flour  or  starch  were  injected. 
He  observed  that  the  small  granules  are  quickly  ingested  by  isolated 
leucocytes,  whilst  the  large  granules  are  surrounded  by  quite  a  layer 
of  phagocytes.  He  agrees  with  several  other  writers,  that  the  amylase 
found  in  defibrinated  blood  has  its  origin  in  leucocytes. 

Leber1,  in  the  course  of  his  researches  on  inflammation,  made  the 
observation  that  the  pus  of  a  hypopyon  that  was  absolutely  aseptic 
digests  coagulated  fibrin  at  a  temperature  of  25°  C.  and  liquefies 
gelatine  very  readily.  Achalme2  has  confirmed  this  and  has  added 
several  other  interesting  data.  He  investigated  the  soluble  ferments 
of  pus  and  directed  his  attention  amongst  others  to  experimental  pus, 
set  up  by  the  injection  of  spirit  of  turpentine.  In  addition  to  amylase 
and  a  ferment  which  liquefies  gelatine,  Achalme  has  discovered  in 
pus,  saponase  (lipase),  casease,  and  a  ferment  closely  allied  to  trypsin. 
This  last  readily  digests  fibrin  and  also  attacks  coagulated  white  of 
egg ;  in  the  products  of  this  digestion  Achalme  found  peptone  but 
could  not  always  obtain  leucin  and  tyrosin.  He  never  succeeded  in 
demonstrating  the  presence  of  sucrase,  inulase,  emulsin  or  lactase 
in  pus.  On  the  other  hand  he  found  large  quantities  of  oxydase, 
thus  confirming  the  discovery  of  Portier3  who  was  the  first  to  demon- 
strate that  these  ferments  met  with  in  the  blood  are,  in  the  living 
animal,  found  inside  leucocytes.  By  a  large  number  of  experiments, 
[103]  carried  out  on  most  diverse  representatives  of  the  animal  kingdom, 
Portier  was  able  to  establish  the  important  fact  that  the  oxydases 
which  are  found  in  many  organs  or  in  the  fluid  of  blood  withdrawn 
from  the  organism  really  originate  in  leucocytes  as  they  deteriorate, 
and  break  up.  In  this  respect,  then,  they  resemble  fibrin-ferment 
very  closely. 

To  complete  the  list,  already  considerable,  of  leucocytic  ferments, 
I  must  further  cite  the  anticoagulating  soluble  ferment  whose 
existence  in  Mammals  has  been  so  well  demonstrated  by  Delezenne. 

All  this  evidence  encourages  us,  then,  to  support  the  thesis  that 
alexine  is  one  of  the  numerous  intraleucocytic  soluble  ferments  and 
that  it  only  passes  into  the  fluids  as  the  result  of  rupture  or  of 
damage  to  the  phagocytes.  Nolf  (I.e.)  has  recently  pronounced 
against  this  view ;  we  must  therefore  examine  his  arguments  closely. 
In  the  first  place  he  takes  his  stand  on  the  analogies  between  the 

1  "Die  Entstehung  der  Entziiudung,"  Leipzig,  1891,  S.  508. 

2  Compt.  rend.  Soc.  de  Biol,  Paris,  1899,  p.  568. 

3  "Les  Oxydases  dans  la  serie  animale,"  Paris,  1897. 


Resorption  of  the  formed  elements  97 

solution  of  the  red  blood  corpuscles  by  the  serums  and  by  certain 
salts.  It  must  not  be  forgotten,  in  connection  with  his  theory,  that 
haemolysis  is  but  one  example,  out  of  many,  of  the  action  of  alexines. 
Of  all  the  formed  elements  the  red  corpuscles  are  the  most  delicate ; 
they  are  readily  broken  up  by  all  sorts  of  agents  (moderate  heat, 
water,  salts,  etc.).  Further,  there  are  numerous  other  cells  (white 
corpuscles,  spermatozoa,  and  inferior  organisms)  which  resist  the 
action  of  salts  much  better,  which,  nevertheless,  are  very  injuriously 
affected  by  the  action  of  the  alexines. 

Nolf  lays  special  stress  on  the  experiments  in  which,  after  keeping 
red  blood  corpuscles  in  prolonged  contact  with  active  serums,  he 
has  looked  in  vain  for  the  peptone  reaction.  He  prepared  his 
mixtures  in  sealed  tubes  or  flasks,  and  kept  them  in  an  incubator  at 
37°  C.  for  24 — 48  hours,  or  even  for  weeks.  Under  these  conditions 
the  haemoglobin  is  transformed  into  metahaemoglobin,  but  peptones 
never  appear.  Nolf  concludes  therefrom  "  with  confidence,  that  the 
alexines  do  not  exert  the  slightest  peptonisiug  effect  on  the  albu- 
minoids of  the  corpuscle  "  (1.  c.  p.  672). 

To  this  conclusion  it  must  be  objected  that  peptone  is  not  the  only 
product  of  the  digestion  of  albuminoids  by  soluble  ferments.  Under 
certain  conditions  the  disintegration  is  carried  much  further,  in 
others  it  is  arrested  at  an  earlier  stage.  Thus  human  urine  which 
contains  pepsin,  never  gives  the  peptone  reaction  with  fibrin ;  the 
digestion  of  the  latter  only  goes  on  up  to  the  stage  of  protalbumose. 
When,  however,  the  urinary  pepsin  is  fixed  on  flakes  of  heated  fibrin  [104] 
which  are  submitted  to  digestion  in  acidulated  water  the  digestion 
proceeds  further  and  gives  as  final  products  deuteroalbumose  and 
peptone1.  Xow,  under  the  conditions  in  Xolf's  experiments  the 
digestion  would  be  very  quickly  stopped,  because,  at  the  temperature 
of  37  C.,  alexine  very  soon  loses  its  strength.  Investigators  who 
have  experimented  with  haemolytic  serums  know  well  that,  even 
when  kept  at  a  low  temperature,  alexine  may  lose  its  activity  within 
24  hours. 

It  has  been  mentioned  above  that  Xolf  sought  in  vain  for 
a  parallel  between  haemolysis  by  salts  and  that  by  serums,  in  what 
relates  to  the  action  of  the  fixative.  He  was  unable  to  find  anything 
comparable  to  this  action  amongst  salts,  although  digestion  by  soluble 

1  Stadelmann,  Ztschr.  f.  Biol,  Munchen,  1887,  Bd.  xxiv,  S.  226 ;  1888,  Bd.  xxv, 
8.  208;  Patella,  Ann.  univ.  di  med.  e  chir.,  Milano,  1887.  (Cited  by  Huppert 
in  Xenbauer  u.  Vogel's  Analyse  des  Hams,  xte  Aufl.,  Wiesbaden,  1898,  S.  599.) 

B.  7 


98  Chapter  IV 

ferments  offers  undoubted  analogies.  I  need  only  recall  further  the 
discovery  of  enterokynase,  the  soluble  ferment  of  the  digestive  juice 
of  the  dog,  which  actively  stimulates  the  action  of  pancreatic  ferments, 
and  especially  that  of  trypsin.  The  recent  researches  of  Delezenne 
(communicated  to  the  International  Congress  of  Physiology  held  at 
Turin  in  September  1901)  support  this  conclusion  in  a  very  im- 
portant fashion.  As  already  pointed  out  in  Chapter  III  the  entero- 
kynase of  the  intestinal  juice  exerts  an  action  comparable  with  that 
of  the  fixatives  of  haemolytic  serums.  Alone,  it  does  not  act  as  a 
solvent  ferment,  but  when  it  attaches  itself  to  the  fibrin  it  aids  the  action 
of  the  trypsin  in  a  marked  degree.  In  pancreatic  digestion  entero- 
kynase plays  the  part  of  the  fixatives  in  the  solution  of  red  corpuscles. 

The  analogy  between  the  resorption  of  formed  elements  and 
intestinal  digestion  extends  even  beyond  this.  When  we  inject,  into 
the  peritoneal  cavity  or  under  the  skin  of  various  animals,  blood  from 
a  different  species,  the  blood  serum  of  the  former  becomes  haemolytic 
for  the  red  corpuscles  of  the  latter.  The  solution  of  these  red 
corpuscles  is  effected  by  the  alexine  of  the  serum,  whose  activity  is 
rendered  very  great  owing  to  the  presence  of  a  quantity  of  specific 
fixative.  This  same  fixative  appears  also  in  the  fluids  of  animals 
to  whom,  instead  of  injecting  blood,  we  simply  give  it  by  the  mouth. 
[105]  This  fact  has  been  established  by  Metalnikoff  \ 

Another  fact  in  favour  of  the  close  relationship  between  the 
fixatives  and  enterokynase  consists  in  the  presence  of  both  in  the 
lymphatic  (lymphopoietic)  organs.  The  fixatives  which  aid  the  solu- 
tion of  red  corpuscles  are  found  specially  in  the  mesenteric  glands. 
Enterokynase,  as  demonstrated  by  Delezenne,  is  found  not  only  in  the 
intestinal  juice,  but  also  in  Peyer's  patches,  the  solitary  glands,  the 
meseuteric  glands,  and  the  leucocytes  of  exudations  and  of  the  blood. 

Supported  by  these  various  facts  we  are  quite  justified  in  regard- 
ing the  haemolysing  substance  of  serum  as  containing  two  soluble 
ferments,  of  which  one,  alexine,  corresponds  to  trypsin,  the  other, 
the  fixative,  resembling  enterokynase.  The  alexine,  whose  nature  is 
gradually  disclosing  itself  with  more  precision,  should  bear  the  name 
of  cytase  or  cell-ferment.  The  cytase  of  the  macrophagic  organs,  or 
macrocytase,  comes  under  this  category.  According  to  the  researches 
of  Tarassewitch  it  also  acts  more  vigorously  when  there  is  added  to 
it  some  of  the  fixative  found  in  the  serum  (heated  to  56°  C.)  of 
prepared  animals. 

1  Centralbl.f.  Bakteriol  u.  Parasitenk.,  Itfl  Abt,  Jena,  1901,  Bd.  xxix,  S.  531. 


Besorption  of  the  formed  elements  99 

We  have  said  that  in  the  living  animal  the  macrocytase  is  localised 
in  the  phagocytes  of  the  organs  and  of  the  blood.  Thus,  when  goose's 
blood  is  injected  into  the  peritoneal  cavity  of  the  guinea-pig  the  red  blood 
corpuscles  are  digested  within  the  macrophage  and  not  in  the  fluid  of 
the  peritoneal  exudation.  When,  however,  the  same  kind  of  blood  is 
injected  a  second  or  a  third  time,  it  is  found  that  a  certain  number  of 
the  red  corpuscles  become  permeable  and  lose  their  haemoglobin,  which 
they  give  up  to  the  fluid  of  the  exudation,  and  only  the  membrane  and 
the  nucleus  remain.  These  are  at  once  ingested  by  the  macrophages 
which  under  these  conditions  manifest  a  real  excess  of  activity.  In- 
stead of  sending  out  small  processes,  as  they  do  after  the  first  injec- 
tion of  blood,  these  phagocytes  move  about  like  true  Amoebae,  sending 
out  broad  pseudopodia,  and  ingest  not  only  the  remains  of  the  red 
corpuscles  but  also  those  still  intact1  (Fig.  20).  Under  these  con- [1 06] 
ditions  macrocytase  must  undoubtedly 
be  found  in  the  peritoneal  plasma 
It  is,  however,  easily  demonstrable  that 
this  ferment  was  not  performed  in  the 
fluid  but  has  escaped  from  the  leuco- 
cytes that  have  undergone  phagolysis. 
After  the  rapid  injection  of  alien  blood 
the  phagocytes  of  the  peritoneal  lymph 
gather  into  clumps,  become  immobile, 
and  for  a  time  lose  their  phagocytic 
power.  It  is  only  after  the  lapse  of  a 
longer  or  shorter  period  that  the  leuco-  Fig  20.-Rapid  ingestion  of 

L  .....  .  red  corpuscles  of  the  goose 

cytes  recover  from  the  phagolysis,  arrive  by  macrophages. 

in  great  numbers  in  the  peritoneal  cavity 
and  display  their  phagocytic  energy. 

If  the  damage  to  the  phagocytes — the  phagolysis — is  the  actual 
cause  of  the  setting  free  of  the  intraleucocytic  ferment,  we  have 
only  to  prevent  this  phagolysis  in  order  to  inhibit  the  solution  of 
red  blood  corpuscles  in  the  fluid  of  the  exudation.  For  this  purpose 
it  is  sufficient  to  prepare  guinea-pigs  (which  have  already  received 
several  injections  of  goose's  blood)  by  means  of  an  injection  of  fresh 
broth,  of  physiological  salt  solution,  or  of  carbonic  acid  into  the 

1  Sawtchenko  (Arch,  russes  de  Path.,  etc.,  St  Petersb.,  1901,  t.  xi,  p.  455)  has 
observed  that  leucocytes,  after  they  have  absorbed  the  specific  fixative,  acquire  the 
property  of  ingesting  red  blood  corpuscles  with  extraordinary  rapidity.  Tarassewitch 
was  able  to  confirm  this  fact. 

7—2 


100  Chapter  IV 

peritoneal  cavity  on  the  eve  of  the  decisive  experiment.  Such  injection 
at  once  provokes  phagolysis,  which  is  then  followed  by  an  abundant 
exudation  of  leucocytes.  When,  next  day,  a  dose  of  red  blood  cor- 
puscles of  the  goose  (deprived  of  serum  by  centrifugalising)  is 
introduced  into  the  peritoneal  cavity  thus  prepared  phagolysis  is  no 
longer  produced,  or  very  feebly,  and  is  of  very  short  duration. 
Under  these  conditions  the  solution  of  the  red  corpuscles  by  the 
peritoneal  fluid  is  reduced  to  a  minimum,  and  in  its  place  an  ex- 
tremely rapid  and  considerable  ingestion  of  red  corpuscles  by  the 
macrophages  may  be  observed.  In  order  that  the  experiment  may 
be  completely  successful  it  is  advisable  to  use  goose's  blood  heated 
to  37°  C.  or  thereabouts  for  the  injection. 

Even  when  the  red  corpuscles  of  the  goose  are  introduced,  not  into 
the  peritoneal  cavity  but  into  the  subcutaneous  tissue  of  guinea-pigs 
that  have  received  several  injections  of  goose's  blood,  we  can  easily 
prevent  the  extracellular  solution  of  the  red  corpuscles  which  takes 
place,  as  already  indicated,  in  the  normal  guinea-pig.  As  in  this 
[107]  case  the  goose's  serum  which  is  mixed  with  the  corpuscles  contributes 
to  the  haemolysis,  it  must  be  suppressed  by  centrifugalising  the 
defibrinated  goose's  blood  and  by  washing  the  corpuscles  with  normal 
saline  solution. 

Collectively,  the  facts  I  have  just  described  clearly  indicate  that 
the  phagocytes  must  be  regarded  as  the  source  of  the  haemolytic 
ferment.  The  macrocytase  remains  in  the  body  of  these  cells  so 
long  as  they  are  in  a  normal  condition ;  but  immediately  they  are 
injured,  in  consequence  of  the  sudden  introduction  of  foreign  sub- 
stances into  the  peritoneal  cavity,  a  portion  of  the  macrocytase  escapes 
and  acts  on  the  red  corpuscles  as  if  it  had  been  employed  in  vitro. 

As  the  conclusion  I  have  just  formulated  is  of  fundamental 
importance  in  the  study  of  resorption  and  immunity  it  is  necessary 
to  support  it  by  as  many  arguments  as  possible.  For  this  reason, 
therefore,  I  feel  obliged  to  draw  the  attention  of  the  reader  to  another 
example  of  the  resorption  of  formed  elements. 

We  have  already  spoken  of  the  resorption  of  spermatozoa  in 
the  peritoneal  cavity,  and  of  the  part  played  by  the  macrophages 
in  this  phenomenon.  As  a  result  of  this  resorption,  just  as  after 
that  of  red  blood  corpuscles,  the  organism  acquires  new  properties 
of  the  same  character.  Landsteiner1  and  the  writer2  have  shown 

1  CentralU.f.  Bakteriol  u.  Parasitenk.,  Ite  Abt,  Jena,  1899,  Bd.  xxv,  S.  546. 

2  Ann.  de  Vlnst.  Pasteur,  Paris,  1899,  t.  xin  p.  738. 


Resorption  of  the  formed  elements  101 

that  the  blood  serum  and  the  peritoneal  fluid  of  animals  that  have 
been  injected  with  the  spermatic  fluid  of  bull,  rabbit,  or  man,  be- 
come spermotoxic,  that  is  to  say,  they  render  the  corresponding 
spermatozoa  motionless  and  kill  them.  These  fluids,  however,  never 
acquire  the  power  of  dissolving,  even  partially,  these  elements. 
The  disappearance  and  final  solution  of  the  spermatozoa  is  only 
effected  within  phagocytes,  and  almost  exclusively  in  the  macro- 
phages. 

Moxter1  has  demonstrated  that  the  spermotoxin  which  appears  in 
the  serum  of  prepared  animals  consists  of  two  substances,  corre- 
sponding to  those  present  in  the  haemolytic  serums.  These  are  the 
macrocytase  (alexine,  complement)  and  the  fixative  (intermediary  or 
sensibilising  substance).  For  him  they  are  identical  with  those  which 
dissolve  the  red  corpuscles.  Without  dwelling  on  the  subject  we 
may  say  that  the  macrocytase  which  dissolves  the  red  corpuscles 
and  that  which  arrests  the  motion  of  the  spermatozoa  are  really 
identical  in  the  same  species  of  animal,  as  is  accepted  and  developed  [108] 
by  Bordet.  On  the  other  hand,  it  is  impossible  to  accept  Moxter 's 
theory  of  the  identity  of  the  two  fixatives.  They  must  be  regarded 
as  different ;  this  we  have  attempted  to  prove  in  one  of  our  memoirs2 
and  is  in  accordance  with  the  law  of  the  specificity  of  fixatives  in 
general. 

The  question  which  interests  us  more  especially  at  this  moment 
is  where  are  these  two  constituent  substances  of  the  spermotoxin 
to  be  found  and  how  do  they  behave  in  the  living  organism  ?  This 
question  has  been  very  thoroughly  studied  by  Metalnikoff3  in  my 
laboratory.  His  experiments  have  been  closely  followed  by  me,  and 
in  presenting  their  principal  results  I  can  bear  witness  to  their 
correctness. 

The  spermotoxin  obtained  by  Metalnikoff  is  distinguished  from 
the  haemotoxins  we  have  discussed  up  to  the  present  in  that  they 
develop,  not  as  a  result  of  the  injection  of  cell  elements  from  a  diffe- 
rent species,  but  as  a  result  of  the  introduction  into  the  organism  of 
spermatozoa  from  the  same  species,  the  guinea-pig.  We  have  here, 
then,  to  deal  with  what  has  been  termed  autospermotoxin. 

The  serum  of  the  normal  guinea-pig  acts  but  feebly  on  the  sper- 
matozoa of  this  species,  which,  under  its  influence,  remain  motile  for 

1  Deutsche  med.  Wchnschr.,  Leipzig,  1900,  S.  61. 

2  Ann.  de  I'Inst.  Pasteur,  Paris,  1900,  t.  xiv,  p.  369. 

3  Ibid.,  p.  577. 


102  Chapter  IV 

several  hours.  When,  however,  guinea-pigs  have  received  one  or 
several  injections  of  the  spermatozoa  of  their  own  species,  their  serum 
and  peritoneal  lymph  become  distinctly  toxic  and  render  the  sperma- 
tozoa motionless  in  a  few  minutes.  In  male  guinea-pigs  so  prepared 
the  serum  acquires  this  toxic  property  not  only  for  the  spermatozoa  of 
other  male  guinea-pigs,  but  likewise  for  those  of  the  individual  itself 
which  furnishes  the  serum.  This  latter,  then,  becomes  distinctly 
autospermotoxic. 

If  the  spermotoxin  were  diffused  in  the  plasma  and  other  fluids  of 
the  guinea-pig  which  furnishes  it,  it  ought  to  render  motionless  the 
spermatozoa  contained  in  the  genital  organs.  Experiment  demonstrates, 
however,  that  this  is  not  the  case.  If  the  male  organs  be  removed 
from  a  guinea-pig  whose  serum  is  very  autospermotoxic  in  vitro,  we 
find,  especially  in  the  epididymis,  a  mass  of  very  virile  spermatozoa 
which  for  a  long  time  retain  their  motility  in  physiological  salt 
[109]  solution.  The  niacrocytase,  then,  has  not  reached  the  spermatozoa 
in  the  living  animal ;  this  is  because  it  is  not  found  in  the  plasmas. 
Let  us  inject  into  a  guinea-pig,  whose  serum  is  strongly  autospermo- 
toxic, one  portion  of  sperm  into  the  subcutaneous  tissue  and  another 
portion  into  the  peritoneal  cavity.  In  the  first  site  a  soft  oedema, 
filled  with  transuded  fluid,  in  which  the  very  active  spermatozoa 
retain  their  motility  for  a  couple  of  hours,  is  produced.  In  the  peri- 
toneal fluid  the  same  spermatozoa  become  motionless  in  a  few 
minutes.  This  great  difference  is  explained  by  the  fact  that,  under  the 
skin,  there  are  no,  or  almost  no  pre-existing  leucocytes,  whilst  in  the 
peritoneal  fluid  they  are  abundant.  The  phagocytes  injured  by  the 
introduction  of  sperm  into  the  peritoneal  cavity,  abandon  a  portion  of 
their  macrocytase,  sufficient  to  render  the  spermatozoa  motionless. 
But  when  Metalnikoff  injected  physiological  salt  solution  into  the 
peritoneal  cavity  of  his  autospermotoxic  guinea-pigs,  and  then,  on  the 
following  day,  a  quantity  of  sperm,  the  spermatozoa  continued  very 
active  for  more  than  an  hour.  In  this  case  phagolysis  is  very  transi- 
tory and  insignificant;  it  is  soon  followed  by  a  great  afflux  of 
leucocytes  which  bring  about  a  rapid  ingestion  of  the  spermatozoa. 
Many  of  these  elements  are  devoured  in  a  living  state  ;  for  even  when 
their  body  is  enclosed  in  the  macrophage,  their  tail,  left  outside, 
continues  to  move  very  actively. 

All  these  experiments  demonstrate  that  in  the  normal  state  the 
macrocytase  remains  within  the  phagocytes  and  only  escapes  during 
phagolysis,  or  at  the  moment  when  the  blood,  after  it  has  been  with- 


Resorption  of  the  formed  elements  103 

drawn  from  the  organism,  coagulates.  Is  it  the  same  for  the  fixative  ? 
It  is  easy  to  prove  that  this  soluble  ferment  circulates  in  the  plasmas 
of  the  living  organism.  We  have  already  said  that  the  spermatozoa 
of  a  guinea-pig  whose  serum  is  very  autospermotoxic,  remain  alive  for 
some  time  in  the  physiological  salt  solution.  But  if  we  introduce 
them,  in  vitro,  into  the  serum  of  a  normal  guinea-pig  they  remain 
motile  but  a  short  time  (some  10 — 20  minutes),  whilst  the  spermatozoa 
of  a  normal  guinea-pig  will  live  in  the  same  serum  for  several  hours. 
This  difference  is  explained  by  the  fact  that  the  spermatozoa  of  the 
autospermotoxic  guinea-pig,  although  very  active,  have  absorbed 
the  fixative  during  the  life  of  the  animal.  This  fixative  is,  as  we 
have  stated,  found  in  the  body  fluids  and  has  been  able  to  penetrate 
to  the  male  organs.  Here  the  spermatozoa  become  charged  with 
the  fixative  and,  once  transported  into  the  serum  of  the  normal  [no] 
guinea-pig,  rich  in  macrocytase,  they  lose  their  movements  very 
quickly.  At  the  same  time  the  spermatozoa  used  for  control,  not 
having  absorbed  any  fixative,  are  able  to  live  for  a  long  time  in  the 
same  serum. 

As  the  macrocytase  remains  fixed  to  the  phagocytes  there  can  be 
no  doubt  as  to  its  origin ;  it  is  elaborated  by  these  cells.  Whence 
however  comes  the  fixative  which  is  free  in  the  body  fluids  and  which 
is  precisely  the  substance  that  is  developed  in  so  large  a  quantity  in 
the  treated  animals  ?  The  exact  solution  of  this  question  is  not  easy  ; 
nevertheless  there  are  many  facts  which  indicate  that  this  fixative  is 
also  of  phagocytic  origin.  We  know  already  that  the  serums  of  normal 
animals  contain  only  small  quantities  or  sometimes,  perhaps,  none  of 
the  fixative.  This  fixative  only  appears  abundantly  as  the  result  of 
the  resorption  of  the  corresponding  elements,  red  corpuscles  or  sper- 
matozoa. This  resorption,  as  we  have  said,  is  almost  exclusively  the 
work  of  the  macrophages.  It  is  just  in  those  cases  where  the  red 
corpuscles,  injected  into  the  peritoneal  cavity  of  an  animal  of  the  same 
species,  pass  directly  into  the  lymph,  without  being  injured  or,  save 
exceptionally,  ingested  by  the  phagocytes,  that  the  fixative  is  not 
formed.  When  the  red  blood  corpuscles  of  the  goose,  introduced  with 
defibrinated  blood  below  the  skin  of  a  guinea-pig,  undergo  there  a 
partial  solution  in  the  fluid  of  the  exudation,  and  where  the  phago- 
cytosis is  more  limited  than  in  the  peritoneal  cavity,  the  production  of 
fixative  is  small.  When  the  injection  of  the  same  goose's  blood  is  made 
into  the  peritoneal  cavity  of  a  guinea-pig  and  is  followed  by  complete 
phagocytosis,  the  fixative  is  produced  in  greater  abundance.  There 


104  Chapter  IV 

exists,  then,  in  all  these  cases  a  constant  relation  between  the  degree 
of  phagocytosis  and  the  amount  of  the  fixative  produced.  As  this 
fixative  facilitates  the  access  of  the  cytase  to  the  cells  and  as  the 
resorption  of  these  elements  takes  place  specially  in  the  macrophages, 
we  are  bound  to  come  to  the  conclusion  that  the  fixative  is  a  second 
phagocytic  ferment  which  is  produced  in  abundance  during  the  process 
of  intracellular  digestion.  Only,  instead  of  remaining  in  the  substance 
of  the  phagocytes,  this  fixative  is  in  part  thrown  out  from  these 
elements.  It  passes  into  the  plasma  of  the  blood  and  into  the  other 
fluids  and  ends  by  disappearing  from  the  organism,  probably  being 
eliminated  by  the  excretory  channels. 

In  the  Invertebrata,  where,  as  we  have  seen,  the  alien  red  blood 
corpuscles  are  also  digested  within  the  phagocytes,  we  have  never 
been  able  to  demonstrate  any  haemolytic  property  of  the  blood  fluid, 
even  after  repeated  injections  of  blood.  We  must  conclude  from  this 
[in]  that  in  these  animals  the  quantity  of  fixative  is  merely  sufficient  to 
bring  about  the  solution  of  the  red  corpuscles  which  are  within  the 
phagocytes.  In  the  case  of  fishes  and  higher  animals  (we  may  recall 
the  example  of  the  red  corpuscles  of  the  guinea-pig  when  resorbed 
into  the  organism  of  the  gold-fish)  the  production  of  the  fixative  is 
much  more  abundant,  and  this  ferment  can  be  easily  demonstrated  by 
its  action  in  vitro. 

This  over-production  of  a  ferment  which  acts  in  the  phagocytic 
resorption,  finds  its  analogue  in  the  passage  of  certain  digestive 
ferments,  such  as  amylase  and  pepsin  in  man  and  the  dog,  into  the 
blood  and  urine,  as  mentioned  in  the  preceding  chapter. 

One  of  the  best  arguments  in  favour  of  the  thesis  here  developed, 
has  been  furnished  to  us  by  the  analysis  of  the  phenomena  observed 
in  connection  with  the  autospermotoxic  serums  of  the  guinea-pig. 
This  idea  of  autotoxins  was  originally  put  forward  by  Ehrlich  in  his 
memoirs,  published  in  conjunction  with  Morgenroth  and  already 
repeatedly  cited.  Ehrlich  asked  himself  whether  the  organism  which 
resorbs,  not  red  corpuscles  of  an  alien  species,  but  red  corpuscles  of 
its  own  species,  would  also  be  capable  of  developing  haemolytic 
substances.  With  this  object  he  injected  blood  obtained  from  goats 
into  these  same  goats  or  into  other  individuals  of  the  same  species. 
He  and  Morgenroth1  were,  under  these  conditions,  able  to  obtain 
isotoxic  serums,  that  is  to  say  serums  which  dissolve  the  red  corpuscles 
of  the  goat,  coming  from  other  individuals  than  those  which  had  been 
1  Berl  klin.  Wchnschr.,  1900,  S.  453. 


Resorption  of  the  formed  elements  105 

treated  by  the  blood  and  which  furnished  the  serum.  In  order  to 
obtain  this  result,  however,  they  had  to  inject,  not  unaltered  blood  but 
blood  mixed  with  water.  The  red  corpuscles  of  the  unaltered  blood 
pass  readily  into  the  circulation  of  the  animal  of  the  same  species, 
without  being  attacked  by  the  phagocytes.  Now,  we  know  from  the 
experiments  of  Bordet  that  the  stromas  of  the  red  corpuscles  suffice 
for  the  production  of  the  fixative,  whilst  the  haemoglobin  does  not 
incite  to  the  development  of  this  ferment  by  the  organism.  As  the 
stromas,  injected  with  a  mixture  of  blood  and  water,  must  be  devoured 
by  the  macrophages,  we  can  readily  understand  that  these  phagocytes 
may  serve  for  the  elaboration  of  the  fixative. 

The  resorption  of  the  red  corpuscles  and  that  of  spermatozoa  which  [112] 
we  have  presented  as  examples,  may  serve  as  types  for  the  resorption 
phenomena  of  formed  elements  in  general.  When  other  species  of 
cells  are  introduced  into  the  organism,  the  resulting  process  always 
reveals  the  same  character  :  inflammatory  reaction  with  preponderant 
intervention  of  the  macrophages ;  intraphagocytic  digestion  of  the 
introduced  elements ;  excessive  production  and  excretion  of  the 
fixatives.  Whilst  the  macrocytase  is  always  the  same  in  the  same 
species  of  animal,  the  fixatives  are  different  and  specific.  In  addition 
to  the  haemofixatives  and  spermofixatives  already  described,  we  may 
obtain,  as  the  result  of  the  injection  of  the  corresponding  cells,  leuco- 
fixatives,  nephrofixatives,  hepatofixatives,  trichofixatives,  etc.  It 
does  not  enter  into  our  programme  to  treat  the  subject  here1.  W^e 
wish  simply  to  insist  on  those  aspects  of  the  resorption  of  cells 
which  are  closely  connected  with  the  problem  of  Immunity.  In  the 
next  chapter  we  must,  however,  recur  to  certain  features  of  the 
phenomena  of  resorption. 

1  We  have  given  a  sketch  of  the  actual  state  of  this  question  of  cell  poisons  or 
cytotoxins  in  the  Revue  generate  des  sciences  pares  et  appliquees,  1901,  p.  1. 


[113]  CHAPTER  V 

RESORPTION  OF  ALBUMINOID  FLUIDS. 

Resorption  of  albuminoid  substances. — The  precipitins  of  blood  serum  which  appear 
as  a  result  of  the  absorption  of  serums  and  of  milk. — Absorption  of  gelatine. — 
Leucocytic  origin  of  the  ferment  which  digests  gelatine. — Antienzymes. — Anti- 
rennet. — The  anticytotoxins. — Antihaemotoxic  serums. — Their  two  constituent 
parts:  anticytase  and  antifixative. — Action  of  anticytase. — The  antispermo- 
toxins. — Origin  of  anticytotoxins. — Ehrlich's  theory  on  this  question. — Origin  of 
antihaemotoxins. — Origin  of  antispermotoxin. — Production  of  this  antibody  by 
castrated  males. — The  antispermofixative  produced  when  the  spermatozoa  are 
excluded.— Distribution  of  spermotoxin  and  antispermotoxin  in  the  organism. 

WE  stated  at  the  beginning  of  the  last  chapter  that  various  fluid 
substances  of  very  complicated  chemical  composition  may  be  absorbed 
by  the  tissues  and  utilised  by  the  organism  without  requiring  to  be 
modified  by  the  digestive  juices  of  the  intestinal  canal.  We  must 
now  describe,  exactly,  the  phenomena  observed  in  these  cases  and 
endeavour  to  establish  the  mechanism  of  the  absorption  of  fluids  in 
the  living  organism. 

We  have  already  cited  the  examples  of  blood  serum,  milk,  and 
white  of  egg,  all  of  which  are  readily  utilised  by  the  organism  which 
receives  them  directly  into  the  peritoneal  cavity  or  below  the  skin. 
The  proof  that  these  substances  are  modified — digested  by  the  tissues, 
is  furnished  by  the  observation  that  their  injection  necessarily 
brings  about  appreciable  changes  in  the  properties  of  the  blood. 
Th.  Tchistovitch1,  in  a  research  carried  out  in  the  Pasteur  Institute, 
was  the  first  to  demonstrate  that  the  resorption  of  the  blood  serums 
of  the  eel  and  horse  by  the  organism  of  the  rabbit,  excites  in  the 
blood  of  the  latter  animal  the  production  of  specific  precipitates. 
The  blood  serum  of  rabbits  that  have  been  vaccinated  against  the 
toxic  eePs  serum  gives  a  precipitate  with  eel's  serum ;  the  serum  of 
rabbits  treated  with  horse's  blood  gives  a  similar  precipitate  with 
[114]  horse's  serum,  etc.  This  property  has  since  been  confirmed  and 

1  Ann.  de  VInst.  Pasteur,  Paris,  1899,  t.  xiu,  p.  413. 


Resorption  of  albuminoid  fluids  107 

studied  by  several  observers,  who  have  made  use  of  it  for  the  recogni- 
tion of  human  blood  in  medico-legal  investigations1. 

Bordet2  has  made  the  discovery  that  intraperitoneal  injections  of 
the  milk  of  cows  into  rabbits  provokes  in  the  blood  serum  of  the 
latter  the  property  of  giving  a  specific  precipitate  with  cow's  milk 
only.  This  precipitation  bears  a  great  resemblance  to  the  coagulation 
of  casein ;  which,  however,  does  not  justify  us  in  identifying  the 
precipitating  substance  with  rennet.  This  fact  has  been  confirmed  for 
several  other  species  of  milk,  and  Schiitze3,  in  an  investigation  carried 
on  in  the  Berlin  Institute,  essayed  to  apply  it  to  the  differentiation 
of  the  various  kinds  of  milk.  In  the  same  order  of  ideas,  researches 
have  been  made  on  the  artificial  precipitins  that  develop  in  the  blood 
as  the  result  of  injection  of  white  of  egg  and  other  albuminoids4. 
Leclainche  and  Valle'e5  have  prepared  animals  in  such  a  fashion  that 
their  serum  produces  a  precipitate  with  urinary  albumen.  The  bio- 
logical precipitin  reactions  are  more  sensitive  than  any  of  the  chemical 
reagents  properly  so  called.  These  specific  substances  in  the  serums 
must  be  looked  upon  as  belonging  to  the  group  of  soluble  ferments, 
approximating  to  the  fixatives  rather  than  to  the  cytases,  since 
they  are  unaltered  by  being  heated  to  56°  C.  Their  action  gradually 
declines  after  passing  60°  C.  but  is  only  destroyed  at  a  temperature 
beyond  70°  C. 

An  analogous  soluble  ferment  has  been  discovered  in  the  blood 
serum  of  animals  treated  with  injections  of  gelatine.  We  owe  to 
Delezenne,  who  has  studied  this  question  in  his  laboratory  at  the 
Pasteur  Institute,  the  most  important  and  most  complete  data  on  the 
resorption  of  gelatine.  The  blood  serum  of  normal  animals  possesses 
only  a  very  feeble  power,  sometimes  even  none,  of  liquefying  gelatine. 
When  however  this  substance  is  injected  several  times,  the  serum,  as 
is  the  rule  for  the  formed  elements  and  quite  a  series  of  fluid  sub- 
stances, acquires  a  much  more  pronounced  activity.  The  gelatine,  [us] 
without  giving  any  precipitate,  is  simply  dissolved  and  will  no  longer 

1  Deutsch,  Oompt.  rend.  XIII  congres  internal,  de  Med.  de  Paris,  and  Centralbl. 
f.  Bacteriol.  u.  Parasitenk.,  Ite  Abt.,  Jena,  1901,  t.  xxix,  S.  661  ;  Uhlenhuth,  Deutsche 
med.  Wchnschr.,  Leipzig,  1901,  S.  82 ;  Wassermann  u.  Schiitze,  Berl.  klin.  Wchnschr., 
1901,  S.  187;  [Nuttall  and  Dinkelspiel,  Journ.  of  Hyg.,  Cambridge,  1901,  Vol.  I, 
p.  367  ;  Nuttall,  Brit.  Med.  Journ.,  London,  1902,  I,  p.  825]. 

2  Ann.  de  rinst.  Pasteur,  Paris,  1899,  t.  xin,  p.  240. 

3  Ztschr.f.  Hyg.,  Leipzig,  1901,  Bd.  xxxvi,  S.  5. 

4  [Myers,    Lancet,    London,    1900,   n,   p.   98,   and   Centralbl.  f.   Bakteriol.   u. 
Parasitenk.,  Ite  Abt,  Jena,  1900,  Bd.  xxvm,  S.  237.] 

5  Compt.  rend.  Soc.  de  biol.,  Paris,  1901,  p.  51. 


108  Chapter  V 

solidify  when  it  is  cooled.  The  ferment  of  the  serum  that  produces 
this  effect  resembles  the  precipitins  in  that  it  withstands  the  action  of 
a  temperature  of  56°  C.  and  is  only  destroyed  beyond  60°  C.  Like  the 
trypsins  it  acts  in  a  weakly  alkaline,  neutral,  or  weakly  acid  medium  ; 
but  digestion  takes  place  best  in  a  slightly  alkaline  medium. 

The  question  of  especial  interest  to  us  is  that  of  the  origin  of  this 
ferment  which  digests  gelatine.  If  several  c.c.  of  a  10  %  solution  of 
this  substance  be  injected  into  the  peritoneal  cavity  of  a  laboratory 
animal,  there  is  provoked  with  certainty,  within  a  few  hours,  a  marked 
leucocytosis  of  the  peritoneal  fluid.  A  considerable  afflux  of  leuco- 
cytes, amongst  which  the  microphages  are  even  more  numerous  than 
the  macrophages,  takes  place.  When  to  a  hanging  drop  of  such  an 
exudation  a  trace  of  Ehrlich's  neutral  red  solution  is  added,  there 
appears  almost  at  once  an  intense  coloration  of  the  numerous  droplets 
inside  the  two  kinds  of  leucocytes.  It  is,  therefore,  manifest  that  the 
gelatine  excites  a  powerful  positive  chemiotaxis  of  the  mobile  phago- 
cytes and  that  it  is  absorbed  by  these  cells.  This  experiment  demon- 
strates that  the  phagocytes  can  not  only  ingest  solid  bodies,  such  as 
the  various  formed  elements,  coloured  granules,  etc.,  but  that  they  are 
also  capable  of  absorbing  fluid  substances  introduced  into  the  tissues 
or  cavities  of  the  organism. 

The  data  brought  forward  by  Delezenne  demonstrate  very  clearly 
the  part  played  by  the  mobile  phagocytes  in  the  digestion  of  gelatine. 
He  obtained  his  best  results  in  the  dog.  We  know  that  it  is  easy  in 
this  animal  to  provoke  an  aseptic  exudation,  very  rich  in  leucocytes. 
This  exudation  when  deprived  of  its  serum  and  washed  with  physio- 
logical salt  solution  gives  a  solution  which  exerts  a  feeble  digestive 
action  on  gelatine.  If  the  exudation  be  produced  in  a  dog  that  has 
previously  received  several  injections  of  this  substance,  we  obtain 
leucocytes  whose  extract,  obtained  by  the  same  method,  will  digest 
gelatine  much  more  actively.  The  digestive  power  of  the  leucocytes 
of  the  treated  dog  is  sometimes  five  times  greater  than  that  of  the 
leucocytes  of  the  normal  dog.  Here,  then,  we  undoubtedly  have  an 
acquired  digestive  power  which  reveals  a  great  reinforcement  of  the 
phagocytic  activity. 

[116]  In  the  prepared  dogs  the  leucocytes  have  a  much  greater  digestive 
action  on  gelatine  than  has  the  blood  serum  of  the  same  animals,  a 
fact  which  indicates  that  the  source  of  the  soluble  ferment  must  be 
sought  for  in  the  phagocytes  themselves.  The  results  of  these 
researches  are  of  great  service  to  us  in  the  study  of  immunity 
properly  so  called. 


Resorption  of  albuminoid  fluids  109 

For  some  time  past  attempts  have  been  made  to  show  that  the 
soluble  ferments,  diastases,  or  enzymes,  are  closely  allied  to  albu- 
minoid substances.  Nencki  and  Mme  Sieber1  support  this  view  by 
their  recent  researches  on  the  chemical  composition  of  pepsin.  In  all 
the  above  cases  there  is  this  in  common  between  the  two  categories  of 
substances,  their  absorption  by  the  organism  is  followed  by  the  appear- 
ance in  the  blood  of  antagonistic  ferments.  Just  as  after  the  injection 
of  milk,  white  of  egg,  serums,  etc.  into  the  cavities  or  tissues,  specific 
precipitins  are  produced,  so  the  injection  of  certain  enzymes  provokes 
the  formation  in  the  organism  of  antienzymes  or  antidiastases. 

It  has  been  known  for  some  time  that  the  blood  serum  of  many 
animals  prevents  the  action  of  certain  enzymes.  Thus  Roden  has 
shown  that  normal  horse's  serum  retards  or  even  completely  prevents 
the  coagulation  .of  milk  by  rennet  It  has  often  been  observed,  too, 
that  normal  serums  hinder,  more  or  less,  the  digestion  of  albuminoids 
by  trypsin.  It  is  only  quite  recently,  however,  that  we  have  begun  to 
prepare  antienzymes  by  the  injection  into  animals  of  corresponding 
enzymes.  Thus,  Hildebrand2  has  succeeded  in  obtaining  an  anti- 
emulsin  in  the  serum  of  rabbits,  into  which  he  had  injected  several 
separate  doses  of  emulsin.  Fermi  and  Pernossi3  have  prepared  an 
anti trypsin,  and  von  Dungern4  has  obtained  an  antidiastase  against  the 
proteolytic  enzymes  of  some  bacteria.  But  of  all  the  antienzymes, 
the  one  that  has  been  best  studied  up  to  the  present  is  indisputably 
antirennet,  obtained  independently  by  Morgenroth5  and  Briot6.  The 
former  of  these  investigators  treated  goats  with  increasing  quantities  [117] 
of  rennet  and  was  able  to  assure  himself,  by  comparative  detailed 
researches,  of  the  appearance  and  increase  in  quantity  of  antirennet 
in  the  blood  serum.  The  goat  which  gave  the  best  result  ceasing  to 
develop  antirennet  it  was  impossible  to  make  the  antirennet  potency 
go  beyond  a  certain  point. 

Briot  also  obtained  antirennet  in  rabbits  into  which  he  had  in- 
jected fluid  rennet  on  several  occasions.  He  was  able  to  convince 
himself  that  the  antirennet  of  horse's  serum  is  a  non-dialysable 

1  Zeitschr.f.physiol  Chem.,  Strassburg,  1901,  Bd.  xxxn,  S.  291. 

2  Virchoic's  Archiv,  Berlin,  1893,  Bd.  cxxxi,  S.  32. 

3  Zeitschr.  f.  Hyg.,  Leipzig,  1894,  Bd.  xvm,  S.  83. 

4  Munchen.  med.  Wchnschr.,  1898,  15  August. 

5  Centralbl  f.  Bakteriol  u.  Parasitenk.,  Ite  Abt.,  Jena,  1899,  Bd.  xxvi,  S.  349, 
and  1900,  Bd.  xxvn,  S.  721. 

6  "Etude  sur  la  presure  et  I'antipresure."    Sceaux,  1900.    (These  d.  L  Faculte  <L 
Sc.  de  Paris,  no.  4.) 


110  Chapter  V 

substance  which  is  precipitated  by  alcohol  and  certain  salts.  Like  the 
precipitins  and  the  diastase  which  digests  gelatine,  antirennet  resists 
a  temperature  of  55°— 56°  C. ;  even  heating  to  58°  C.  has  no  effect  on 
the  antirennet  serum.  At  60°  C.,  however,  the  heat  begins  to  exert  an 
injurious  effect,  and  after  three  hours  at  62°  C.  the  serum  has  lost 
all  power  to  prevent  the  coagulation  of  the  casein  by  antirennet. 
Morgenroth  and  Briot  both  state  that  the  antirenuet  neutralises  the 
rennet  by  a  direct  action. 

The  cell  poisons,  or  cytotoxins,  of  animal  origin  which  were 
treated  in  the  preceding  chapter,  likewise  set  up  the  production  of 
special  anti-bodies,  or  anticytotoxins.  The  consideration  of  these  latter 
has  a  very  special  interest  for  those  who  study  the  question  of  immunity 
from  a  general  point  of  view.  The  first  discovery  of  these  anticyto- 
toxins was  made  in  connection  with  the  study  of  the  toxic  power  of  the 
blood  serum  of  eels.  Camus  and  Gley1  and,  independently  of  them, 
H.  Kossel2  demonstrated  that  animals  when  treated  with  increasing 
doses  of  eel's  serum  acquire  an  antitoxic  property  which  protects 
their  corpuscles  against  the  haemolytic  action  of  ichthyotoxin,  or  the 
toxic  substance  of  the  blood  of  eels.  Th.  Tchistovitch3  has  not  only 
confirmed  this  discovery,  but  has  added  to  it  new  and  interesting  data. 
When  antitoxic  serum  is  mixed  in  vitro  with  red  blood  corpuscles 
of  the  species  which  furnished  the  serum  and  there  is  added  to  it 
some  haemolytic  eel's  serum,  it  will  be  found  that  the  red  corpuscles 
remain  quite  unaltered.  In  the  control  tubes,  however,  in  which  the 
antitoxic  serum  is  replaced  by  normal  serum  of  the  same  species,  the 
red  corpuscles  are  very  readily  dissolved  under  the  toxic  influence  of 
[118]  the  eel's  serum.  In  animals  (rabbits)  that  are  treated  with  this  latter 
fluid,  there  is  established  not  only  an  antitoxic  power  of  the  blood, 
but  the  red  corpuscles  acquire  a  resisting  power  more  or  less  pro- 
nounced against  the  ichthyotoxin  of  eel's  serum.  When  the  red 
corpuscles  are  separated  from  the  serum  of  rabbits  (treated  with  eel's 
serum)  and  some  ichthyotoxin  is  added  to  them,  solution  very  often 
does  not  take  place  at  all.  According  to  the  experiments  of 
Tchistovitch  there  is  no  direct  relation  between  this  acquired  re- 
sistance and  the  antitoxic  power  of  the  blood.  Sometimes  even  a 
kind  of  antagonism  is  observed  between  the  two  properties  ;  that  is  to 
say,  the  red  corpuscles  of  a  rabbit  whose  serum  is  very  antitoxic  may 

1  Arch,  internal,  de  Pharmacodyn.,  Bruxelles  et  Paris,  1898,  t.  in  and  iv. 

2  Berl.  klin.  Wchnschr.,  1898,  S.  152. 

3  Ann.  de  I'lnst.  Pasteur,  Paris,  1899,  t.  xm,  p.  406. 


Resorption  of  albuminoid  fluids  III 

be  extremely  sensitive  to  the  poison  of  the  eel,  whilst  the  converse 
may  also  hold  good  [cf.  infra,  p.  120]. 

The  toxic  action  of  the  eel's  serum  upon  the  red  corpuscles  of  a 
great  number  of  Vertebrates  is  a  natural  property  which  demands  no 
previous  treatment  of  the  eel.  It  is  the  antitoxic  power,  directed 
against  the  ichthyotoxin,  which  is  developed  only  as  a  result  of  the 
preparation  of  the  animals  by  the  administration  of  increasing  doses 
of  eel's  serum.  Nevertheless  we  also  find  natural  antitoxins  present 
in  the  blood  of  man  or  animals  that  have  not  been  treated  and  which 
act  against  the  cell  poisons,  cytotoxins,  so  widely  distributed  in  the 
blood  of  a  large  number  of  species  of  animals. 

Besredka1  has  demonstrated  that  the  blood  serum  of  Man  and 
many  Vertebrates  contains  a  substance  which  prevents  the  solution  of 
red  corpuscles  under  the  influence  of  blood  serums  of  a  different 
species.  To  reveal  the  presence  of  these  antitoxins  it  is  useful  to  heat 
the  serums  to  56°  C.  and  then  to  add  to  them  red  corpuscles  of  the 
same  species  and  some  haemolytic  serum  of  a  different  species. 
Under  these  conditions  the  solution  of  the  red  corpuscles  does  not 
take  place,  whilst  their  mixture  with  haemolytic  serum  alone,  in- 
evitably provokes  haemolysis. 

Along  with  these  natural  antihaemolysins  there  exist  a  number  of 
artificial  antihaemolysins  or  antihaemotoxins.  Jules  Bordet2  was  the 
first  to  draw  attention  to  this  important  subject.  He  first  obtained 
these  antihaemolysins  by  injecting  blood  serum  of  the  fowl,  which 
possesses  a  very  great  haemolytic  power  on  the  red  corpuscles  of  the 
rabbit,  into  individuals  of  this  latter  species.  After  some  injections,  [119] 
the  serum  of  these  treated  rabbits  was  found  to  be  antihaemotoxic 
against  the  fowl's  serum.  Later3,  Bordet  obtained  a  serum  against  an 
artificial  haemotoxin.  The  serum  of  the  guinea-pig  is  innocuous  to  the 
red  corpuscles  of  the  rabbit.  But  when  rabbit's  blood  was  injected 
several  times  into  guinea-pigs  the  serum  of  the  latter  became  very 
solvent  for  the  red  corpuscles  of  the  rabbit.  To  prevent  this  action  it 
is  sufficient  to  inject  the  haemotoxin  of  treated  guinea-pigs  several 
times  into  rabbits.  The  serum  of  these  rabbits  becomes  antihaemo- 
toxic and  protects  the  red  corpuscles  of  the  rabbit  against  the  solvent 
action  of  guinea-pig's  serum. 

In  the  normal  haemolytic  serums,  such  as  the  serums  of  the  eel  and 

1  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t  xv,  p.  785. 

*  Hid.  1899,  t.  xm,  p.  285. 

3  Ibid.,  Paris,  1900,  t.  xiv,  p.  270. 


112  Chapter  V 

fowl,  the  presence  of  two  substances  which  act  by  combining  could 
not  be  demonstrated.  On  the  other  hand,  in  the  serums  that  were 
obtained  as  a  result  of  the  treatment  of  animals  by  the  injection  of 
blood  from  a  different  species,  it  was  easy  to  demonstrate,  as  we  have 
shown  in  the  preceding  chapter,  the  presence  of  two  constituent 
substances  which  are :  the  macrocytase  (alexine,  complement)  and 
the  fixative  (amboceptor  of  Ehrlich,  sensibilising  substance  of  Bordet). 
For  this  reason  the  study  of  the  antihaemotoxins  obtained  against 
artificial  haemotoxins  is  endowed  with  special  interest.  As  the  solu- 
tion of  the  red  corpuscles,  in  this  case,  can  be  prevented  either  by  an 
antitoxic  action  directed  against  the  cytase,  or  by  a  neutralisation  of 
the  fixative  (for  the  concurrence  of  these  two  substances  is  indis- 
pensable in  order  that  the  solution  may  take  place),  Bordet  asked 
whether  the  antitoxic  serum,  obtained  by  him  in  rabbits,  is  anticytatic 
or  antifixative,  or  whether  it  contains  both  properties.  Before  re- 
solving this  problem  it  was  necessary  to  establish  some  of  the 
essential  characters  of  artificial  antihaemotoxic  serums.  The  principal 
one  amongst  them  is  the  resistance  of  these  antihaemotoxins  to  a 
temperature  of  55 — 60°  C. ;  even  when  heated  to  70°  C.  the  antihaemo- 
toxins retain,  at  least  in  part,  their  fundamental  property.  In  this 
respect  these  substances  differ  from  the  cytases  and  approach  the 
precipitins,  fixatives  and  agglutinins. 

The  very  exact  experiments  carried  out  by  Bordet  have  demon- 
strated that  in  the  serum  of  rabbits,  treated  with  the  specific 
[120]  haemotoxic  serum  of  guinea-pigs,  two  substances,  an  anticytase  and 
an  antifixative,  are  found  in  combination.  The  former  of  these 
antitoxins  is  found  in  abundance,  but  the  amount  of  antifixative  is 
very  small.  Bordet  was  led  to  this  result  in  the  following  way.  To 
prevent  the  solution  of  the  red  corpuscles  of  the  rabbit  in  the  haemo- 
toxic serum  of  the  guinea-pig,  it  was  necessary  for  him  to  add  a 
considerable  dose  (10  to  20  times)  of  the  antitoxic  serum.  When, 
however,  he  heated  the  latter  to  55°  C.  the  quantity  of  this  serum 
necessary  to  prevent  haemolysis  could  be  reduced  very  considerably. 
In  place  of  its  being  necessary  to  add  to  the  haemotoxic  serum  10  or 
20  volumes  of  antitoxic  serum,  it  was  sufficient  to  add  three  or 
sometimes  only  two  volumes  of  this  heated  serum.  As  we  know 
already,  heating  to  55°  C.  destroys  the  macrocytase  which  should  be 
found  in  the  antitoxic  blood  of  the  rabbit.  This  cytase  by  itself  is 
incapable  of  dissolving  the  red  corpuscles  of  the  same  species  ;  but 
when  it  is  added  to  the  fixative  of  the  haemotoxic  serum  of  the 


Resorption  of  albuminoid  fluids  113 

guinea-pig  the  macrocytase  of  the  rabbit's  serum  dissolves  them  very 
readily.  Hence  the  conclusion  that  in  the  haemotoxic  serum  of  the 
guinea-pig  there  must  be  present  a  quantity  of  fixative  sufficient  to 
allow  of  the  solution  of  the  red  corpuscles  by  the  macrocytase  of  the 
rabbit's  serum.  This  antitoxic  serum,  therefore,  which  only  prevents 
the  haemolysis  on  the  condition  of  being  added  in  comparatively 
large  quantity,  contains  very  little  antifixative.  When,  by  heating 
this  serum  to  55°  C.  we  destroy  the  rabbit's  macrocytase,  the  mixture 
of  antitoxic  serum  of  the  rabbit  and  haemotoxic  serum  of  the  guinea- 
pig,  which  ordinarily  dissolves  the  red  corpuscles  of  the  rabbit,  now 
leaves  them  intact.  The  reason  is  that  the  free  fixative  contained 
in  this  mixture  does  not  find  any  available  macrocytase :  that  of  the 
rabbit  being  destroyed  by  the  heating,  and  that  of  the  guinea-pig 
neutralised  by  the  antitoxic  serum.  The  experiment  I  have  just 
described  proves  that  this  antitoxic  serum  contains  specific  anticytase. 
This  anticytase  is  capable  of  neutralising  the  guinea-pig's  macrocytase, 
but  is  altogether  powerless  against  that  of  the  rabbit.  This  last  cir- 
cumstance allows  us  to  investigate  whether  the  antitoxic  serum  of 
the  rabbit  contains,  in  addition  to  anticytase,  a  specific  antifixative. 
Bordet  prepared  a  mixture  of  antitoxic  serum  of  the  rabbit,  heated 
to  55°  C.,  with  haemotoxic  serum  of  the  guinea-pig,  also  heated  to 
55°  C.  In  this  mixture  the  two  macrocytases  (that  of  rabbit  and  that 
of  guinea-pig)  have  been  destroyed  by  heat,  but  the  antitoxins  of  the 
rabbit's  serum  and  the  fixative  of  the  haemotoxic  serum  have  re- 
mained intact.  This  mixture  owing  to  its  want  of  macrocytases  was  [121] 
incapable  of  dissolving  the  red  corpuscles  of  the  rabbit.  By  adding 
to  it  some  fresh  unheated  serum  from  a  normal  rabbit  the  rabbit's 
macrocytase  was  introduced.  As  the  latter  could  not  be  neutralised 
by  the  anticytase  of  the  antitoxic  serum  and  was  incapable,  by  itself, 
of  dissolving  the  red  corpuscles  of  the  rabbit,  it  was  unable  to  produce 
haemolysis  except  on  the  condition  that  there  is  in  the  mixture  a 
sufficient  quantity  of  unneutralised  free  specific  fixative.  As  a 
matter  of  fact,  the  red  corpuscles  of  the  rabbit  are  not  dissolved  in 
the  mixture  described;  this  proves  that  the  fixative  had  become 
inactive  in  consequence  of  the  presence  of  an  antifixative  in  the 
antitoxic  serum  of  the  rabbit.  I  need  not  enter  into  further  details 
of  Bordet's  experiments,  which  have  fully  demonstrated  the  fact  that 
in  the  antitoxic  serum  of  his  rabbits  there  were  really  two  antitoxins  ; 
an  anticytase  abundant  in  quantity,  and  an  antifixative  present  in 
much  smaller  amount. 

B.  8 


114  Chapter  V 

Ehrlich  and  Morgenroth1  quite  independently  of  Bordet  have 
shown  that  an  antihaeinotoxic  serum  is  very  rich  in  anticytase.  After 
making  a  number  of  injections  of  normal  horse's  serum  (very  rich  in 
cytase)  into  a  goat,  they  obtained  in  the  blood  serum  of  the  latter  an 
anticytase  very  active  against  the  cytase  of  the  horse.  This  antitoxic 
serum  of  the  goat,  as  might  be  anticipated,  contains  no  antifixative, 
the  horse's  serum  that  served  for  the  injections  coming  from  normal 
horses  which  contained  no,  or  very  little,  fixative.  Even  in  another 
case,  where  these  investigators2  injected  a  dog  with  sheep's  serum 
very  rich  in  fixative  specific  for  the  red  corpuscles  of  the  dog,  they 
did  not  succeed  in  obtaining  any  antifixative.  These  observations  in 
no  way  diminish  the  value  of  the  discovery  of  the  antifixative  by 
Bordet,  though  they  demonstrate  that  this  antitoxin  cannot,  in 
certain  cases,  be  found  in  the  serum.  Ehrlich  and  Morgenroth  them- 
selves throw  out,  in  this  connection,  the  suggestion  that  in  these 
cases  the  antifixative  remains  linked  to  the  cell  which  produces  it, 
without  being  thrown  off  into  the  blood. 

The  very  precise  data  that  we  have  just  summarised  do  not  seem 
to  agree  with  the  statements  of  certain  other  investigators.  Thus 
[122]  Schlitze3,  from  his  researches  on  the  antihaemotoxic  serum  of  guinea- 
pigs,  directed  against  the  rabbit's  haemotoxin,  has  arrived  at  the 
conclusion  that  in  the  former  an  antifixative  only  is  produced.  As 
he  merely  injected  into  his  guinea-pigs  haemotoxic  rabbit  s  serum 
that  had  been  heated  to  60°  C.  and  consequently  deprived  of  the 
macrocytase,  he  concluded  that  in  this  serum  there  remained  only 
the  specific  fixative  capable  of  provoking  the  formation  of  an  anti- 
toxin. This  must  consequently  be  an  antifixative.  Paul  Miiller4  came 
to  a  similar  conclusion,  after  injecting  rabbits  with  the  heated  hae- 
motoxic serum  of  fowls.  These  injections  caused  the  formation  in  the 
rabbit's  serum  of  an  antitoxin  that  Miiller  regarded  as  an  antifixative. 

Ehrlich  and  Morgenroth5  objected  to  this  interpretation,  taking 
their  stand  on  experiments  made  with  the  serums  of  normal  animals. 
They  were  able  to  show  that  these  serums,  when  injected  in  the  fresh 
state  or  after  being  heated  to  60°  C.,  caused  the  production  of  a  corre- 
sponding antihaemotoxin  which  is  nothing  but  an  anticytase.  When 

1  Her  1.  klin.    Wchnsclir.,   1900,  S.    684.     Ehrlich,   "Croonian   Lecture,"  Proc. 
jRoy.  Soc.  London,  1900,  Vol.  LXVI,  p.  424. 

2  Berl.  klin.  Wchnschr.,  1901,  S.  570. 

3  Deutsche  med.  Wchnschr.,  Leipzig,  1900,  S.  431. 

4  Centralbl.f.  Bakteriol.  u.  Parasitenk.,  Ite  Abt.,  Jena,  1901,  Bd.  xxix,  S.  175. 
6  Berl.  klin.  Wchnschr.,  1901,  S.  251. 


Resorption  of  albuminoid  fluids  115 

Schutze  and  Paul  Miiller  concluded  that  by  heating  tlie  serums  they 
had  entirely  deprived  them  of  cytase  elements  they  did  not  take  into 
account  the  possibility  of  the  cytases  being  transformed,  under  the 
influence  of  heat,  into  other  bodies  unable  to  produce  haemolysis,  but 
quite  capable  of  provoking  the  formation  of  anticytases.  Ehrlich  and 
Morgenroth  give  to  these  new  bodies,  derived  from  cytases  under 
the  influence  of  temperatures  between  55° — 60°  C.,  the  name  of 
complemcntoids ;  and  these  complementoids  appear  in  the  experiments 
of  Schutze  and  Miiller  to  have  caused  the  production  of  antitoxins — 
anticytases. 

In  all  the  investigations  just  summarised  the  anticytases  have  been 
obtained  by  the  injection  into  animals  of  various  blood  serums,  fresh  or 
heated.  Wassermann1  has  discovered  another  method  of  arriving  at 
the  same  result.  He  injected  into  guinea-pigs  the  leucocytes  of 
rabbits,  carefully  deprived  of  all  traces  of  serum.  After  some  time 
the  blood  serum  of  guinea-pigs  thus  treated  became  weakly  but 
distinctly  anticytatic.  From  this  experiment  Wassermann  draws  the 
conclusion  that,  as  has  been  often  affirmed  by  several  observers, 
the  leucocytes  really  contain  cytases. 

How  do  the  anticytases  act  upon  the  cytases  ?  On  this  point  all  [123] 
observers  who  have  studied  this  question  have  but  one  answer,  the 
action  of  the  anticytases  is  direct.  Bordet  thinks  that  the  two  sub- 
stances combine  so  intimately  that  they  cannot  be  again  separated  by 
heat.  We  know  that  the  cytases  are  very  sensitive  to  heat  and  that 
their  haemolytic  property  is  destroyed  at  55°  C.  The  anticytases,  on 
the  other  hand,  as  already  noted,  are  much  more  resistant  to  the  action 
of  heat.  Bordet  has  prepared  mixtures  of  haemolytic  cytase  serum 
and  of  antihaemolytic  serum,  neutral  mixtures,  that  is  to  say,  inactive 
for  red  corpuscles  or  with  a  very  feeble  action  upon  red  corpuscles 
that  have  been  sensibilised  by  the  specific  fixative.  These  mixtures  no 
longer  exhibit  antihaemotoxic  properties  or  they  exercise  this  power 
in  a  very  feeble  degree.  If  in  these  mixtures  the  cytases  remain 
uncombined  alongside  the  anticytases,  it  is  to  be  expected  that  heat- 
ing them  to  55°  C.  will  restore  the  antihaemotoxic  function  of  the 
anticytases ;  the  cytases  being  destroyed  at  55°  C.  there  will  remain 
in  the  mixtures  only  active  anticytase.  The  experiments  made  on 
this  point  have  demonstrated  that  the  heating  of  these  mixtures  does 
not  restore  the  antihaemotoxic  action,  that  is  to  say,  the  anticytase  is 
definitely  combined  with  the  cytase. 

1  Ztschr.f.  Hyg.,  Leipzig,  1901,  Bd.  xxxvn,  S.  190. 

8—2 


116  Chapter  V 

Ehrlich  and  Morgenroth  have  satisfied  themselves  that  their  anti- 
haemotoxin  exerts  no  influence,  either  upon  the  red  corpuscles  or 
upon  the  fixative,  and  is  only  capable  of  preventing  the  action  of  the 
cytase.  They  introduced  red  corpuscles  of  the  rabbit  into  a  mixture 
of  goat's  serum,  heated  to  56°  C.  and  thus  only  retaining  its  fixative, 
and  anticytase  serum.  The  fluid  bathing  the  red  corpuscles  was  then 
removed  by  centrifugalisation  and  the  corpuscles  were  mixed  with 
normal  haemolytic  horse's  serum.  Solution  of  the  red  corpuscles 
took  place  at  once  as  the  anticytase  had  been  completely  removed 
during  centrifugalisation,  being  combined  with  neither  the  red 
corpuscles  nor  the  fixative. 

These  investigators  have  obtained  various  anticytases  by  injecting 
serum  of  various  species  of  animals  into  other  mammals.  They  ob- 
served, however,  that  injections  of  the  serum  of  an  allied  species  did 
not  bring  about  the  formation  of  anticytases.  Thus  the  injection  of 
goat's  serum  into  sheep,  or  of  that  of  sheep  into  goats,  never  produced 
anticytase  serum. 

In  addition  to  antihaemotoxic  serums  several  other  analogous 
[124]  anticytotoxic  serums  have  now  been  obtained.  Thus  Delezenne  has 
prepared  serums  which  prevent  the  action  of  neurotoxin  and  of  the 
cell  poison  which  destroys  the  liver  cells.  We1  have  been  able  to 
obtain  a  rabbit's  serum  which  prevents  the  spermatozoa  of  this  rodent 
being  rendered  motionless  by  the  specific  spermotoxin  of  the  guinea- 
pig.  More  recently  Metalnikoff2,  working  in  my  laboratory,  has 
prepared  another  antispermotoxic  serum  which  prevents  the  specific 
spermotoxiu  of  the  rabbit  from  arresting  the  movement  of  the 
guinea-pig's  spermatozoa. 

As  the  history  of  these  antispermotoxins  presents  certain  interest- 
ing general  features  we  may  with  advantage,  perhaps,  dwell  on  some 
of  their  characters.  The  two  antispermotoxins  mentioned  above  are 
distinguished  by  certain  peculiarities.  When  Metalnikoff  set  to  work 
to  inject  rabbit's  spermotoxin  into  guinea-pigs,  he  thought  that  he  had 
an  easy  task  before  him  and  that  after  a  few  injections  the  guinea- 
pig's  serum  would  become  antispermotoxic.  This,  however,  was  not 
the  case.  The  serum  from  these  animals  when  mixed,  with  spermotoxic 
serum  was  powerless  to  prevent  the  immobilisation  of  the  spermatozoa 
of  the  guinea-pig.  It  was  only  when  he  heated  the  serum  of  his  treated 
guinea-pigs  to  56°  C.  that  the  antispermotoxic  power  appeared  with 

1  Ann:  de  Vlnst.  Pasteur,  Paris,  1900,  t.  xiv,  p.  5. 

2  Ibid.,  p.  583. 


Resorption  of  albuminoid  fluids  117 

the  greatest  distinctness.  The  inefficacy  of  the  unheated  serum  must 
therefore  depend  on  the  toxic  action  of  the  guinea-pig's  macrocytase, 
because  it  is  this  substance  alone  that  can  have  been  destroyed  by  the 
heating  process.  Now,  in  order  that  this  macrocytase  may  act,  the 
presence  of  the  fixative  is  necessary,  which  leads  us  to  the  conclusion 
that  the  serum  of  the  guinea-pigs  injected  by  MetalnikofF  contained 
no  antifixative.  This  hypothesis  was  fully  confirmed  by  experiment. 
Metalnikoff  introduced  a  drop  of  guinea-pig's  serum  into  a  mixture  of 
aiitispermotoxic  serum,  heated  to  56°  C.,  with  spermotoxic  serum. 
The  spermatozoa  continued  their  movements  in  normal  fashion.  But 
when  afterwards  he  added  a  few  drops  of  unheated  serum  from  a 
normal  guinea-pig  the  motions  of  the  spermatozoa  were  arrested 
almost  instantaneously.  Consequently  there  was  present  in  the  mix- 
ture rabbit's  macrocytase  which  had  been  neutralised  by  the  anticytase 
of  the  prepared  guinea-pig's  serum  and  for  that  reason  the  spermatozoa 
remained  motile.  But  in  the  same  mixture  we  had  also  the  specific  [125] 
fixative,  coming  from  the  rabbit's  spermotoxic  serum,  which  remained 
free  and  not  neutralised.  The  motile  spermatozoa  had  become  im- 
pregnated with  this  fixative  and  a  little  guinea-pig's  macrocytase 
(against  which  the  auticytase  was  powerless)  was  sufficient  to  make 
them  suddenly  cease  their  movements. 

There  is  no  doubt,  then,  that  the  serum  of  guinea-pigs  that  have 
been  treated  with  spermotoxin  contains  anticytase  only  and  no, 
or  almost  no,  antifixative.  Such  is  not  the  case  with  the  antispermo- 
toxin  obtained  by  us  in  rabbits  that  were  treated  with  spermotoxic 
toxin  of  guinea-pigs.  Several  consecutive  injections  were  sufficient  to 
render  the  serum  of  the  rabbits  so  treated  capable  of  preventing  the 
action  of  the  spermotoxic  serum  of  the  guinea-pig  on  the  motility  of 
the  rabbit's  spermatozoa.  In  the  mixture  of  antispermotoxic  serum 
and  spermotoxic  serum  these  spermatozoa  continue  to  move  for  a 
considerable  time,  whilst  in  the  control  mixture  prepared  with  normal 
rabbit's  serum  and  spermotoxic  serum  they  become  motionless  at  the 
end  of  a  few  minutes.  To  obtain  this  marked  effect  it  was  not 
necessary  to  heat  the  antispermotoxic  serum  as  in  MetalnikofFs  case. 
Indeed  I  have  performed  almost  all  my  experiments  with  fresh  serums, 
unheated.  As  the  rabbit's  serum  contains  macrocytase  capable  of 
rendering  the  spermatozoa,  sensibilised  by  the  fixative,  motionless 
and  as  this  macrocytase  cannot  be  neutralised  by  the  anticytase  that 
is  active  against  the  guinea-pig's  macrocytase,  the  fact  I  have  just 
pointed  out  indicates  that  the  antispermotoxic  serum  of  my  rabbits 


118  Chapter  V 

contains  antifixative.  The  difference  between  the  antispcrmotoxic 
serum  obtained  by  Metalnikoff  and  that  prepared  by  me  is  similar 
to  that  observed  between  the  antihaemotoxic  serums.  Some  contain 
only  anticytase  but  others  undoubtedly  contain  antifixative  also. 

As  this  result  appeared  to  me  to  be  of  far-reaching  importance  I 
felt  bound  to  verify  it  by  another  method.  I  injected  certain  rabbits 
with  spermotoxic  serum  of  the  guinea-pig  and  others  with  normal 
guinea-pig's  serum.  The  amount  of  cytases  being  about  the  same  in 
both,  the  strength  of  the  serums  obtained  as  the  result  of  injections 
of  normal  serum  and  of  specific  serum  should  be  the  same  if  the 
antispermotoxic  serums  contain  anticytase  only.  Experiment  demon- 
strates just  the  contrary.  The  autispermotoxic  serum  of  rabbits 
treated  with  normal  guinea-pig's  serum  was  on  every  occasion  much 
[126]  less  active  than  the  serum  of  rabbits  injected  with  the  spermotoxic 
serum  of  prepared  guinea-pigs.  The  former  contains  anticytase  only, 
whilst  the  latter  contains  in  addition  antifixative.  Weichhardt's1 
experiments  carried  out  in  my  laboratory  corroborated  the  con- 
clusion I  have  just  formulated. 

Having  made  ourselves  acquainted  with  the  constitution  of  the 
auticytotoxins  we  may  now  pass  to  the  question  of  the  origin  of  these 
bodies  and  of  analogous  ferments  which  act  in  the  resorption  of 
albuminoid  substances  in  the  blood  and  in  the  tissues. 

We  have  already  mentioned  that  the  leucocytes  are  charged  with 
a  soluble  ferment  which  digests  gelatine,  and  that  in  animals  treated 
with  injections  of  gelatine  these  cells  elaborate  a  much  larger  amount 
of  the  ferment.  Here  we  have  evidence  of  a  kind  of  education  of  the 
leucocytes  to  produce  a  greater  amount  of  digestive  ferment,  in  a 
manner  quite  analogous  to  that  which  has  been  described  in  Chapter 
III  in  connection  with  the  augmentation  of  the  pancreatic  ferments 
in  intestinal  digestion.  It  is,  then,  quite  permissible  to  look  upon 
leucocytes,  and  probably  phagocytes  in  general,  as  the  source  of  the 
soluble  ferment  that  digests  gelatine. 

Is  this  the  case  with  the  other  substances  which  take  an  active 
part  in  the  resorption  of  albuminoid  substances  in  the  fluids  and 
tissues  of  the  organism  ?  Up  to  the  present  the  origin  of  precipitins 
and  antiferments,  such  as  antirennet,  has  not  been  studied.  The 
problem  being  very  complex  and  difficult,  it  appears  to  be  impossible 
at  present  to  solve  it.  It  is  known  indeed  that  the  introduction  of 
these  substances  into  the  organism  provokes  a  reaction  similar  to  the 
1  Ann.  de  Plnst.  Pasteur t  Paris,  1901,  t.  xv,  p.  833. 


Resorption  of  albuminoid  fluids  119 

one  we  have  described  in  the  case  of  the  injection  of  gelatine  into  the 
peritoneal  cavity  of  guinea-pigs.  Thus  Morgenroth x  observed  that  in 
his  goats  the  subcutaneous  injection  of  sterile  rennet  caused  the 
formation  of  extensive  infiltration  at  the  seat  of  inoculation,  this 
being  accompanied  by  fever ;  we  are  justified  in  concluding  from  this 
that  rennet  provokes  a  marked  leucocytic  reaction.  Hildebrandt2  has 
demonstrated  by  direct  experiment  that  rennet,  when  enclosed  in 
capillary  glass  tubes  and  introduced  below  the  skin  of  rabbits,  induces 
a  marked  positive  chemiotaxis.  This  led  to  the  formation  of  a  leuco- 
cytic plug  several  millimetres  long.  Now  we  know  from  Briot  that  [127] 
the  rabbit  is  capable  of  producing  antirennet.  Hildebrandt  has  further 
shown  that  several  other  diastases,  or  hydrolytic  ferments,  such  as 
sucrase  and  emulsin,  give  rise  to  a  similar  chemiotactic  phenomenon. 
The  leucocytic  reaction  is  consequently  a  general  phenomenon  following 
the  introduction  into  the  tissues  of  substances  of  complex  chemical 
composition  capable  of  provoking  the  formation  of  antibodies.  We 
are  tempted  from  this  fact  to  accept  it  as  a  law  that  the  leucocytes  are 
capable  of  producing  these  latter  substances.  Although  this  hypo- 
thesis may  be  very  probable,  the  number  of  facts  at  our  disposal  is  not 
yet  sufficient  to  justify  the  statement  that  its  truth  is  demonstrated. 

Since  it  is  the  red  corpuscles  which  are  affected  by  the  haemotoxins 
it  might  be  asked  whether  it  may  not  be  that  these  elements  defend 
themselves  by  the  production  of  antihaemotoxins  the  overplus  of 
which  is  thrown  into  the  blood  and  fluids  in  general  ?  The  researches 
that  have  been  made  on  this  point  relate  especially  to  the  antihaemo- 
toxin  of  the  blood  serum  of  rabbits  in  relation  to  the  ichthyotoxin  of 
eeFs  serum. 

We  must  therefore  examine  the  collected  evidence  bearing  on 
anticytotoxins  and  analogous  bodies  and  endeavour  to  form  some  idea 
as  to  their  probable  origin.  A  large  accumulation  of  exact  data  bear- 
ing on  the  antihaemotoxins  does  not  afford  us  sufficient  information  as 
to  the  source  of  these  substances. 

Let  us  first  examine  the  question,  is  it  possible  to  attribute  to  the 
red  corpuscles  the  function  of  producing  the  antihaemotoxins?  If 
these  elements  are  really  the  source  of  the  antihaemotoxins  it  is 
probable  that  the  red  corpuscles  of  animals  whose  serum  is  anti- 
haemotoxic  will  exhibit  marked  resistance  to  the  toxins ;  thus  we 
know  that  the  white  corpuscles  which  produce  gelatinase  digest 

1  Centralblf.  Bakteriol  u.  Parasitenk.,  Ite  Abt.,  Jena,  1899,  Bd.  xxvi,  S.  352. 

2  Vir chow's  Archiv,  Berlin,  1893,  Bd.  cxxxi,  S.  5. 


120  Chapter  V 

gelatine  much  better  than  does  the  serum  of  the  same  animals.  From 
the  experiments  of  Tchistovitch  (1.  c.  supra  p.  110)  on  rabbits  that 
have  been  immunised  against  eel's  ichthyotoxiu,  it  must  be  accepted 
that  the  red  corpuscles  of  these  animals  are  often  very  sensitive  to 
the  action  of  the  poison  at  a  period  when  the  blood  serum  of  the 
same  rabbits  exhibits  a  marked  antihaemotoxic  power.  It  is  not 
until  later  in  the  process  of  immunisation,  when  the  serum  loses  a 
great  part  of  this  power,  that  the  red  corpuscles  become  resistant  to 
the  ichthyotoxin. 

But  before  we  abandon  the  hypothesis  of  the  production  of  anti- 
haemotoxins  by  the  red  corpuscles  we  must  see  if  it  cannot  be 
reconciled  with  the  facts,  by  the  application  of  Ehrlich's  side-chain 
[128]  theory1.  This  theory  was  evolved  with  the  object  of  explaining  the 
production  of  antitoxins  and  their  action  on  bacterial  and  vegetable 
toxins.  Later,  Ehrlich  has  extended  it  to  the  cytotoxins,  anticyto- 
toxins  and  bactericidal  substances. 

According  to  Ehrlich  the  complex  molecule  of  albuminoid  sub- 
stances contains,  besides  the  central  stable  nucleus,  a  number  of 
side-chains,  or  "receptors,"  which  fulfil  various  accessory  functions 
and  serve  especially  for  the  nutrition  of  the  cell.  These  receptors 
have  a  great  affinity  for  the  various  substances  necessary  for  the  main- 
tenance of  the  life  of  the  cell.  Under  normal  conditions  these  receptors 
seize  nutritive  molecules,  as  a  leaf  of  Dionaea  seizes  the  fly  that 
serves  it  as  food.  Under  special  conditions  these  receptors  lay  hold 
of  complex  molecules  of  albuminoid  substances,  such  as  the  various 
toxins.  In  this  case  the  receptor,  instead  of  combining  with  a  molecule 
which  supports  life,  fixes  a  molecule  which  poisons  the  cell.  Accord- 
ing to  Ehrlich's  theory  on  the  constitution  of  toxins  their  molecules 
contain  an  atomic  group  which  poisons — the  toxophore,  and  another 
group  which  combines  with  the  receptor— the  haptophore.  The  toxic 
group  of  a  complex  poison,  such  as  ichthyotoxin,  cannot  penetrate  into 
a  red  corpuscle  except  by  the  help  of  the  haptophore  group  and  of  the 
corresponding  receptor.  When  a  red  corpuscle  has  absorbed  a  large 
number  of  molecules  of  ichthyotoxin,  the  united  action  of  the  toxo- 
phore groups  renders  life  impossible  and  the  corpuscle  is  dissolved. 
But  when  a  red  corpuscle  has  been  touched  by  only  a  few  toxic 
molecules,  too  few  to  compromise  life,  there  is  merely  immobilisation 

1  Klin.  Jahrb.,  Jena,  1 897,  Bd.  vi,  S.  299 ;  "  Croonian  Lecture,"  Proc.  Roy.  Soc.  Lon- 
don, 1900,  Vol.  LXVI,  p.  424.  Ehrlich,  Lazarus  u.  Pincus,  "Leukaemie,  etc."  in  Nothnagel's 
Specielle  Pathologic  u.  Therapie,  Wien,  1901,  Bd.  vm,  Schlussbetrachtungen,  S.  163. 


Resorption  of  albuminoid  fluids  121 

of  the  receptors  which  are  combined  with  the  haptophore  groups  of 
the  ichthyotoxin.  As  these  receptors  fulfil  an  important  function  in 
the  nutrition  of  the  red  corpuscles,  the  latter  reproduce  them  in  larger 
numbers  than  were  originally  present.  We  know  that  in  the  pheno- 
mena of  repair  an  over-production  of  the  new-formed  parts  often 
takes  place  and,  according  to  Ehrlich,  to  this  over-production  the  pre- 
sence of  antitoxins  in  the  fluids  of  the  body  is  due.  The  receptors, 
developed  in  excess  by  the  red  corpuscles,  fill  these  cells,  and  no 
longer  finding  room  therein  are  extruded  from  them  and  overflow 
into  the  blood  and  other  fluids  of  the  organism.  When  a  fresh  injec-  [129] 
tion  of  toxin  makes  its  way  to  the  blood  it  there  meets  with  a  number 
of  free  receptors,  endowed  with  an  affinity  for  the  haptophore  group 
of  the  molecule  of  the  toxic  substance.  The  chemical  combination 
between  the  two  substances  takes  place  at  once  in  the  plasmas,  a  fact 
which  prevents  the  haptophore  group  of  the  toxin  from  uniting  with 
the  receptor  of  the  red  corpuscles  and  so  injuring  these  cells  by  in- 
troducing the  toxophore  group  into  them.  According  to  this  theory 
the  same  receptors  which,  in  the  free  state  in  the  fluids,  fulfil  the 
antitoxic  function  become  in  the  interior  of  the  red  corpuscles  the 
vehicles  of  intoxication  and  consequently  fulfil  a  philotoxic  function. 
This  opposite  role  of  the  receptors  has  often  been  compared  to  a 
lightning-conductor;  so  long  as  the  receptors  are  attached  to  the 
molecule  of  the  living  protoplasm  they  attract  the  toxin  just  as  a 
lightning-conductor  attracts  the  lightning  when  it  is  badly  insulated. 
So  interpreted,  it  is  easy  to  conceive  that  the  red  corpuscles  of 
animals  whose  fluids  are  antihaemotoxic  may  be  sensitive  to  the 
toxic  action  of  eel's  serum,  as  has  been  observed  by  Tchistovitch.  As 
soon  as  the  protective  fluids  have  been  removed  from  the  red  cor- 
puscles of  the  immunised  organism,  the  corpuscles  when  placed  in 
contact  with  ichthyotoxin  (eel's  serum)  attract  the  haptophore  groups 
of  the  poison  by  means  of  their  numerous  receptors.  These  hapto- 
phores  in  their  turn  introduce  the  toxophore  groups  which  dissolve 
the  red  corpuscles  without  the  slightest  difficulty.  This  theory  does 
not  explain  the  cases,  which  are  numerous,  in  which  the  red  corpuscles 
of  rabbits  that  are  vaccinated  against  eel's  poison  resist  this  poison. 
Camus,  Gley,  and  Kossel,  working  independently,  have  arrived  at  the 
result  that  the  red  corpuscles  of  immunised  rabbits,  from  which  the 
serum  has  been  carefully  removed,  are  not  dissolved  when  submitted 
to  the  action  of  ichthyotoxin,  whilst  the  red  corpuscles  of  untreated 
rabbits  placed  under  the  same  conditions,  undergo  a  rapid  solution. 


122  Chapter  V 

Tchistovitch  confirming  this'  fact  has  added  to  it  the  observation  that 
the  resistance  of  the  red  corpuscles  of  the  rabbit  is  most  often  found 
when  the  serum  loses  its  antitoxic  power.  If  the  receptors  of  the 
red  corpuscles  of  immunised  rabbits,  owing  to  their  great  affinity 
for  the  haptophore  group  of  the  ichthyotoxin  molecule,  only  attract 
the  toxophore  group  of  this  poison,  as  the  lightning-conductor  when 
badly  insulated  attracts  the  lightning,  the  red  corpuscles  should 
[130]  never  manifest  resistance.  To  explain  this  contradiction  we  must  not 
suppose  that  the  red  blood  corpuscles  which  have  become  resistant 
have  got  rid  of  their  receptors.  In  fact,  if  these  receptors  are  so 
necessary  to  the  nutrition  of  the  cell  that  their  absence  has  set  up 
this  extraordinary  over-production  which  has  inundated  the  fluids,  it 
is  evident  that  one  cannot  admit  the  existence  of  red  corpuscles 
entirely  deprived  of  corresponding  receptors. 

When  examined  from  different  points  of  view  the  hypothesis  of 
the  production  of  antihaemotoxin  by  the  red  corpuscles  is  surrounded 
with  very  great  difficulties.  It  appears  to  be  probable,  therefore,  that 
the  source  of  this  antitoxin  must  be  sought  for  in  other  cell  elements, 
and  we  may  be  allowed  to  recall  to  mind  those  cells  which  manifest  a 
general  and  local  reaction  of  the  most  constant  kind  after  each  in- 
jection of  ichthyotoxin.  Tchistovitch  has  observed  that  eel's  serum 
when  introduced  into  rabbits  in  non-fatal  but  immunising  doses 
excites  a  marked  hypeiieucocytosis. 

The  question  of  the  origin  of  anticytotoxins  being  so  complicated, 
it  has  been  necessary  for  its  elucidation  to  seek  an  experimental 
method  of  excluding  the  organ  in  which  the  antibody  is  supposed  to 
have  its  origin.  As  we  cannot  think  of  eliminating  the  red  or  white 
corpuscles,  nor  the  greater  part  of  the  tissues  and  organs,  there 
remains  only  one  way  of  bringing  about  this  result.  It  is  the  sup- 
pression of  the  male  genital  organs.  We  know  already  that  the 
injection  of  semen  readily  excites  the  production  of  a  spermotoxin, 
and  that  this  spermotoxin  gives  rise  to  the  development  of  a  cor- 
responding antispermotoxin.  If  it  is  the  spermatozoa,  that  is  to  say 
the  elements  having  a  particular  affinity  for  the  spermotoxin,  which 
elaborate  the  antitoxin  we  must  conclude  that  castrated  males  would 
be  incapable  of  producing  it.  With  this  in  view  we  have  carried  out  a 
great  number  of  experiments  which  have  amply  proved  to  us  that  male 
rabbits  when  deprived  of  their  sexual  organs  are  fully  as  capable  of 
developing  antispermotoxin  in  their  fluids  as  are  control  rabbits 
in  which  the  male  genital  apparatus  remains  intact.  Doe-rabbits, 


Resorption  of  albuminoid  fluids  123 

and  young,  sexually  immature  rabbits  of  both  sexes,  also  react  to 
injections  of  spermotoxin  by  producing  the  corresponding  antispermo- 
toxin.  The  specific  elements  which  are  sensitive  to  the  action  of  a 
cytotoxin  undoubtedly  are  not  indispensable  for  the  development  of 
the  corresponding  anticytotoxin.  This  result  is  in  complete  harmony 
with  the  hypothesis  above  put  forward,  that  the  red  corpuscles  cannot  [131] 
be  regarded  as  the  source  of  the  antihaemotoxin.  In  the  case  of  anti- 
spermotoxin  this  fact  can  be  rigorously  established  by  experiment. 

Here  arises  the  following  question.  We  have  seen  that  the  anti- 
cytotoxins  are  composed  of  two  different  substances :  an  anticytase 
and  an  antifixative.  The  former  is  an  antitoxin  capable  of  neutralising 
macrocytase,  the  soluble  ferment  which  will  attack  indifferently  all 
kinds  of  cell  elements.  It  is  not  to  be  wondered  at,  then,  that  the 
exclusion  of  the  spermatozoa  in  no  way  prevents  the  production  of 
anticytase  by  an  organism  which  receives  injections  of  cytotoxins. 
These  latter,  as  we  have  already  said,  contain  cytase  along  with  the 
specific  fixative ;  the  macrocytase  can  attack  any  kind  of  animal  cell 
provided  that  it  can  find  some  fixative  or  any  other  means  to  penetrate 
into  the  interior  of  these  formed  elements.  We  have  seen  that  the 
antispermotoxin,  obtained  by  Metalnikoff  in  guinea-pigs,  does  not 
contain  any  anticytase.  Amongst  his  animals  treated  with  spermo- 
toxin was  a  castrated  male  guinea-pig  which  also  produced  anticytase. 
There  is  nothing  astonishing  in  this  fact,  the  injected  cytase  must  have 
linked  itself  to  many  other  cells  which  were  able  to  develop  anticytase. 

But  the  example  of  the  antispermotoxin  of  the  rabbits  in  my  own 
experiments  is  very  different.  In  order  that  it  might  manifest  its  action 
the  serum  of  these  rabbits  did  not  need  to  be  heated  to  56°  C. ;  it  was 
not  necessary  to  rid  it  of  its  own  macrocytase  which  could  have  acted 
under  the  influence  of  the  fixative,  if  this  latter  for  want  of  antifixa- 
tive had  remained  free  in  the  added  spermotoxin,  This  autifixative, 
then,  is  undoubtedly  found  in  the  serum  of  castrated  males  which  have 
shown  themselves  capable  of  producing  not  only  anticytase,  but  also 
antifixative.  This  result  has  been  further  verified  by  comparative 
experiments  on  castrated  male  rabbits,  some  of  which  received 
spermotoxic  guinea-pig's  serum  whilst  the  others  received  only 
normal  guinea-pig's  serum.  It  has  been  demonstrated  that  the 
amount  of  cytases  remains  almost  constant  in  both  normal  and 
vaccinated  animals1.  If,  then,  the  antispermotoxins  contain  only 

1  Bordet,  Ann,  de  Vlnst.  Pasteur,  Paris,  1895,  t.  ix;  p.  499;  von  Dungern, 
Munchen.  med.  Wchnschr.,  1900,  S.  678. 


124  Chapter  V 

[132]  anticytase,  the  injection  of  specific  guinea-pig's  serum  and  that  of 
normal  guinea-pig's  serum  should  produce  the  same  result,  that  is 
to  say  the  serums  of  castrated  rabbits,  when  treated  by  these  two 
kinds  of  guinea-pig's  serum,  should  exhibit  the  same  antispermotoxic 
power.  Experiments  have,  however,  proved  that  this  is  not  the  case. 
The  serum  of  castrated  rabbits  that  have  been  injected  several  times 
with  normal  guinea-pig's  serum  becomes  distinctly  autispermotoxic, 
but  its  power  to  protect  the  spermatozoa  of  the  rabbit  against 
being  deprived  of  motility  by  the  guinea-pig's  spermotoxin  is  greatly 
inferior  to  that  which  is  developed  in  the  serum  of  other  castrated 
rabbits  that  I  injected  with  spermotoxic  guinea-pig's  serum.  Of 
course  all  the  other  conditions  of  the  experiment  were  the  same 
for  the  two  groups  of  rabbits. 

Several  series  of  facts,  then,  focus  to  this  fundamental  point,  that 
the  organism  of  an  animal  that  has  been  deprived  of  its  male  sexual 
organs  is  in  a  condition  to  produce  antispermofixative.  Against  the 
argument  that  we  have  drawn  from  the  fact  that  the  antispermotoxic 
serum  of  castrated  rabbits  that  have  been  treated  with  spermotoxic 
serum  acts  without  being  heated,  might  be  cited  certain  experiments 
made  by  Ehrlich  and  Morgenroth.  The  antispermotoxic  action  in  this 
case,  as  already  stated,  demonstrates  that  the  serum  of  prepared 
rabbits  contains  antifixative.  Otherwise,  had  the  fixative  not  been 
neutralised,  it  would  have  allowed  the  macrocytase  of  the  rabbit's 
serum  to  arrest  the  movements  of  the  spermatozoa.  Now  the  two 
above-named  observers  have  demonstrated1  that  the  injection  of 
different  serums  into  animals  is  capable  of  exciting  in  their  blood  the 
development  of  anticytases.  The  macrocytase  of  castrated  rabbits 
which,  before  treatment  with  the  spermotoxin,  was  capable  of  arresting 
the  movements  of  rabbits'  spermatozoa  acted  upon  by  a  fixative, 
might  become  inert  after  the  injections  of  spermotoxic  serum  of 
guinea-pigs.  To  clear  up  this  point  I  asked  M.  Weichardt2,  who  has 
carried  out  work  on  this  subject  in  my  laboratory,  to  try  by  means 
of  unheated  serums  of  normal  animals,  to  restore  the  activity  of 
spermotoxin  that  had  been  mixed  with  antispermotoxic  serum.  Sper- 
matozoa of  rabbits  were  put  into  a  definite  mixture  of  spermotoxic 
guinea-pig's  serum,  heated  to  56°  C.,  and  antispermotoxic  serum,  also 
heated  to  56°  C.,  obtained  from  castrated  rabbits  that  had  been  treated 
with  spermotoxin.  The  spermatozoa  remained  very  active  in  this 

1  Bert.  klin.  Wchnschr.,  1901,  S.  255. 

2  Ann.  de  TInst,  Pasteur,  Paris,  1901,  t.  xv,  p.  833. 


Resorption  of  albuminoid  fluids  125 

mixture  which  contains  specific  fixative  (in  the  spermotoxic  guinea-  [133] 
pig's  serum)  and  antispermotoxiii.  To  this  mixture  is  added  a  little 
normal  rabbit's  or  horse's  serum,  unheated.  These  serums  contain 
cytases  and  would  be  quite  capable  of  arresting  the  movements  of  the 
spermatozoa  if  there  was  found  in  the  mixture  any  free  fixative  that 
would  enable  the  macrocytase  to  be  linked  to  the  spermatozoa.  Under 
these  conditions  the  spermatozoa  remain  motile  for  a  long  time.  The 
fixative,  then,  was  no  longer  active  ;  it  was  neutralised  by  the  anti- 
fixative  of  the  antispermotoxic  serum  of  castrated  rabbits.  A  control 
experiment  was  made  with  the  same  substances ;  but  the  castrated 
rabbits'  serum  that  had  been  treated  with  spermotoxic  serum  was 
replaced  by  the  serum  of  other  castrated  rabbits  treated  with  normal 
guinea-pig's  serum.  In  these  latter  mixtures  the  spermatozoa  became 
motionless  at  the  end  of  a  very  short  time ;  the  fixative,  not  being 
neutralised,  readily  allowed  the  rabbit's  and  horse's  cytases  to  affect 
the  spermatozoa. 

It  follows  from  all  this  that  the  antispermotoxic  serum  of  castrated 
male  rabbits,  when  treated  with  normal  guinea-pig's  serum,  contains 
anticytase  only  ;  whilst  the  serum  of  castrated  male  rabbits,  treated 
with  specific  and  spermotoxic  guinea-pig's  serum,  contains  anticytase 
and  antifixative.  The  latter,  then,  has  been  produced  independently 
of  the  sensitive  elements, — the  spermatozoa. 

Having  established  the  fact  that  antispermotoxin  does  not  come 
from  the  male  organs,  it  was  necessary  to  try  to  ascertain  its  true 
source.  With  this  object  in  view  we  injected  spermotoxic  serum  into 
young  rabbits  (quite  capable  of  producing  antispermotoxin)  and  tried 
to  follow  the  fate  of  the  spermotoxin  in  the  organism.  When  spermo- 
toxic guinea-pigs  serum  is  injected  into  the  peritoneal  cavity  of  the 
rabbit  a  notable  amount  of  spermotoxin  is  found  in  the  thickened 
portion  of  the  omentum  made  up  of  lymphoid  tissue.  But  the  greater 
portion  of  the  poison  passes  into  the  circulation  whence  it  goes  to  fix 
itself  in  various  organs,  especially  the  spleen.  At  the  moment  when 
the  spermotoxin  is  found  in  the  blood  a  certain  quantity  of  this  fluid 
was  drawn  off  into  tubes  containing  some  drops  of  extract  of  leeches' 
heads.  After  the  blood  thus  treated  had  been  centrifugalised  the 
plasma  was  decanted  and  its  power  of  arresting  the  movements  of 
spermatozoa  was  compared  with  that  of  serum  of  the  same  blood 
prepared  in  the  usual  way.  From  these  researches  it  results  that  the 
plasma  is  always  richer  in  spermotoxin  than  is  the  corresponding  [134] 
serum.  Sometimes  the  difference  in  favour  of  the  plasma  is  very  great. 


126  Chapter  V 

A  part  of  the  spermotoxin  passes  into  the  kidneys  and  the  supra- 
renal capsules.  It  is  probable  that,  as  is  the  case  with  so  many  soluble 
poisons,  a  certain  proportion  of  the  spermotoxin  may  be  eliminated 
by  the  uropoietic  organs.  A  small  quantity  of  this  poison  is  found 
also  in  the  male  and  female  sexual  glands  of  young  non-castrated 
rabbits. 

The  search  for  some  main  centre  of  origin  for  the  production  of 
antispermo toxin  has  as  yet  led  to  no  positive  result.  The  power  of 
arresting  the  movements  of  spermatozoa  first  appears  in  the  blood 
plasma,  and  it  is  this  same  fluid  which,  later,  is  more  antispermotoxic 
than  is  any  organ.  Amongst  the  tissues  which  fix  spermotoxin  the 
genital  organs  play  not  the  slightest  part  in  the  production  of  anti- 
spermotoxin.  The  experiments  with  castrated  rabbits  afford  sufficient 
proof  of  this.  On  the  other  hand  it  becomes  more  and  more  probable 
that  the  phagocytic  system,  disseminated  in  many  organs,  and  especially 
the  leucocytes,  furnish  the  antispermotoxic  substance.  The  fixation 
of  the  spermotoxin  by  the  leucocytes  of  the  blood,  such  as  the  cells  of 
the  omentum  and  of  the  spleen,  already  offers  us  a  valuable  indication. 
The  absence  of  any  particular  organ  that  might  have  the  monopoly  of 
fixing  the  spermotoxin  and  which  should  later  be  found  charged  with 
a  predominant  amount  of  antispermotoxin  also  speaks  in  favour  of  the 
phagocytic  origin  of  this  antitoxin. 

After  a  single  intraperitoneal  injection  of  spermotoxic  guinea-pig's 
serum  into  young  rabbits,  the  blood  of  the  latter  is  distinctly  spermo- 
toxic for  several  days ;  later  it  becomes  indifferent,  but  eight  or  ten 
days  after  the  commencement  of  the  experiment  the  blood  begins  to 
exhibit  an  antispermotoxic  power.  In  these  cases  the  plasma  shows 
itself  more  active  than  the  serum.  When  the  rabbits  are  killed  at  this 
stage  of  commencing  antitoxic  production,  it  is  found  that  an  extract 
of  the  organs  is  not  antispermotoxic  or  only  feebly  so.  In  all  cases  this 
power,  when  it  exists,  is  more  feeble  than  that  of  the  blood  fluid.  The 
results  obtained  with  extracts  of  organs  are  not  constant.  Sometimes 
the  spleen  possesses  more  antitoxic  activity,  whilst  the  liver,  thymus, 
omentum,  lymphatic  glands  and  genital  glands  exhibit  none  of  this 
property.  In  other  cases  the  survival  of  the  spermatozoa  that  are 
[135]  influenced  by  the  spermotoxin  has  been  longest  in  the  extract  of  the 
suprarenal  capsules.  Sometimes  the  extract  of  the  omentum  exhibits 
the  greatest  antispermotoxic  power.  This  great  variability  in  the 
development  of  the  property  of  protecting  the  spermatozoa  accords 
well  with  the  idea  that  the  elements  which  produce  antispermotoxin 


Resolution  of  albuminoid  fluids  127 

are  wandering  cells  which,  under  diverse  influences,  may  be  localised 
in  very  diverse  points  of  the  organism. 

We  must  not  deceive  ourselves.  The  facts  which  have  been 
collected  up  to  the  present  do  not  allow  us  as  yet  to  form  a  final 
opinion  on  the  origin  of  auticytotoxins,  but  we  are  quite  justified  in 
regarding  as  very  probable  the  hypothesis  that  the  phagocytes  play  a 
most  important  part  in  the  process.  It  is  in  all  cases  beyond  doubt 
that  the  amoeboid  cells  which  resorb  the  formed  elements  play  a  very 
important  part  in  the  resorption  of  fluids  of  very  complex  molecular 
composition. 


128 


[136]  CHAPTER  VI 

NATURAL  IMMUNITY  AGAINST  PATHOGENIC 
MICRO-ORGANISMS 

Natural  immunity  and  the  composition  of  the  body  fluids. — Cultivation  of  the 
bacteria  of  influenza  and  pleuro-pneumonia  in  the  fluids  of  refractory  animals. — 
Resistance  of  Daphniae  to  the  Blastomycetes. — Examples  of  natural  immunity 
in  Insects  and  Mollusca. — Immunity  of  Fishes  against  the  anthrax  bacillus. — 
Immunity  of  frogs  against  anthrax,  Ernst's  bacillus,  the  bacillus  of  mouse 
septicaemia  and  the  cholera  vibrio. — Natural  immunity  in  the  cayman. — 
Immunity  of  the  fowl  and  pigeon  against  anthrax  and  human  tuberculosis. — 
Immunity  of  the  dog  and  rat  against  the  anthrax  bacillus. — Immunity  of 
Mammals  against  anthrax  vaccines. — Immunity  of  the  guinea-pig  against 
spirilla,  vibrios,  and  streptococci. — Natural  immunity  against  anaerobic  bacilli. 
— Fate  of  Blastomycetes  and  Trypanosomae  in  the  refractory  organism. 

IN  the  third  chapter  reference  has  been  made  to  the  frequency  of 
cases  of  natural  immunity  against  infective  diseases.  Examples  of  this 
immunity  occur  in  the  lower  animals — the  Invertebrata — and  are 
widely  met  with  among  the  Vertebrata.  We  have  already  mentioned 
that  this  natural  immunity  can  be  attributed  neither  to  insuscepti- 
bility to  microbial  toxins  nor  to  the  elimination  of  the  micro- 
organisms by  the  excretory  channels.  Nevertheless  the  pathogenic 
agents  which  have  penetrated  into  the  tissues  of  the  refractory 
organism  disappear,  without  being  eliminated.  To  facilitate  the 
study  of  their  disappearance  it  has  been  necessary  to  pass  in  review 
the  phenomena  that  follow  the  introduction  of  foreign  bodies  into 
the  organism  and  to  present  a  brief  analysis  of  the  process  of  re- 
sorption  of  cell  elements  in  its  relations  to  digestion.  We  have  tried 
to  demonstrate  that  resorption  is  nothing  more  than  a  process  of 
digestion  which,  instead  of  going  on  in  the  intestinal  canal,  takes  place 
in  the  tissues ;  that  it  is,  indeed,  an  intracellular  digestion  exactly 
comparable  to  that  which  serves  for  the  nutrition  of  certain  of  the 
lower  animals. 


Immunity  against  pathogenic  miwo-organisms    129 

A  knowledge  of  all  these  facts  is  necessary  before  we  can  deal 
with  the  subject  to  which  the  present  chapter  must  be  devoted — 
the  innate  natural  immunity  of  animals  and  man  against  pathogenic  [137] 
micro-organisms.  As,  under  natural  conditions,  it  is  the  micro- 
organism and  not  its  toxic  products  which  invades  the  organism,  it 
is  clear  that  we  must  give  the  first  place  to  the  study  of  immunity 
against  the  micro-organism.  The  more  so  because  this  form  of  im- 
munity is  much  more  frequently  met  with  than  is  an  insusceptibility 
to  toxins. 

Since  the  animal  organism  has  a  very  variable  composition  it 
might  be  concluded  that  the  micro-organisms  find  in  the  refractory 
species  simply  a  chemical  medium  in  which  they  cannot  live.  We 
cannot  go  far  in  the  discussion  of  this  supposition  without  seeing  that 
it  may  be  rejected.  Among  the  pathogenic  micro-organisms  some  are 
distinguished  by  a  great  fastidiousness  and  sensitiveness  as  regards 
the  medium  in  which  they  are  placed.  Such,  for  example,  are  the 
parasites  of  malaria  and  their  allies.  They  live  inside  the  red  blood 
corpuscles  of  Vertebrata  and  appear  to  be  extremely  discriminating 
in  regard  to  their  requirements.  All  animals,  even  monkeys,  are 
refractory  to  human  malarial  fevers.  It  might  be  concluded  from 
this  that  here  at  least  the  immunity  may  be  due  to  the  fact  that  the 
chemical  composition  of  the  contents  of  the  red  corpuscles  in  the 
immune  animals  is  different  from  that  of  the  red  corpuscles  of  man. 
But  when  we  see,  as  was  first  demonstrated  by  Ross1,  that  the  malaria 
parasite  of  Laveran,  having  made  its  way  into  the  digestive  canal 
of  certain  mosquitos  (Anopheles),  there  develops  abundantly,  it  is 
difficult  to  maintain  this  thesis. 

Among  other  micro-organisms  of  animal  origin  we  have  the  Try- 
panosoma,  the  parasite  of  the  terrible  disease  propagated  by  the 
Tsetse  fly  which  commits  such  ravages  amongst  mammals.  Man  alone 
escapes  it,  exhibiting  a  natural  immunity  that  nothing  apparently  can 
overcome.  Are  we  to  affirm  that  it  is  the  difference  in  the  chemical 
composition  of  the  human  body  which  assures  to  man  his  immunity 
against  a  parasite  that  attacks  indifferently  an  herbivorous  animal, 
such  as  the  ox  or  rabbit,  or  a  carnivorous  animal,  such  as  the  dog  ? 
In  these  examples  I  have  chosen  merely  those  micro-organisms  which 
it  has  never  been  possible  to  cultivate  on  any  artificial  nutrient 

1  Brit.  Mecl  Journ.,  London,  1897,  n,  p.  1786;  1898, 1,  p.  550.     Ann.  de  VInsL 
Pasteur,  Paris,  1899,  t.  xm,  p.  136. 

B.  9 


130  Chapter  VI 

medium  and  which  are  kept  alive  with  very  great  difficulty  outside 
the  living  organism. 

What  is  to  be  said  then  of  the  vegetable  micro-organisms  which,  in 
[13S]  this  respect,  are  much  less  exacting  ?  The  most  important  of  these 
and  the  most  numerous  of  all  pathogenic  micro-organisms,  the 
Bacteria,  can  as  a  rule  be  cultivated  without  difficulty  not  only  in 
the  blood  and  fluids  of  animals  that  are  susceptible  or  refractory 
to  their  morbific  action,  but  also  on  all  kinds  of  vegetables  and 
artificial  media :  broths,  fluids  composed  of  mineral  salts  and  of 
certain  organic  substances.  It  is  really  not  possible  to  attribute  the 
natural  immunity  of  the  dog  and  the  fowl  against  the  anthrax 
bacillus — so  fatal  to  a  great  number  of  mammals,  man  included, — 
to  its  incapacity  to  feed  on  the  fluids  of  these  animals,  when  we  see 
that  this  same  bacillus  is  capable  of  killing  lower  animals,  such  as 
the  cricket,  and  can  thrive  on  carrots,  potatoes  and  other  vegetables. 

Even  when,  among  the  bacteria,  we  take  those  that  are  most 
exacting  in  the  choice  of  their  food,  we  still  find  it  impossible  to 
explain  natural  immunity  as  being  due  to  the  want  of  power  on  the 
part  of  these  organisms  to  obtain  their  nutriment  from  the  juices 
of  refractory  species.  The  bacillus  discovered  by  R  Pfeifler1  in 
influenza  does  not  develop  on  media  that  are  ordinarily  employed  in 
bacteriology  in  the  cultivation  of  a  great  number  of  micro-organisms. 
It  needs  a  special  food,  which  is  prepared  for  it  by  spreading  a  drop 
of  fresh  blood  on  the  surface  of  agar.  Pfeifler  has  established  the 
fact — confirmed  by  many  observers — that  the  best  species  of  blood 
to  use  for  this  purpose  is  that  of  the  pigeon.  We  should  have  to 
believe,  then,  did  the  immunity  really  depend  on  the  composition 
of  the  fluids,  that  the  pigeon  is  the  least  refractory  of  all  animals. 
Experiment  has  demonstrated  the  erroneousness  of  such  a  supposi- 
tion :  the  pigeon  is  quite  as  refractory  to  Pfeiffer's  bacillus  as  are 
most  other  species  of  animals. 

As  a  second  example  the  bacterium  of  bovine  pleuro-pneumonia 
may  be  cited.  It  is  the  smallest  of  all  known  bacteria.  The  diffi- 
culties surrounding  the  discovery  and  identification  of  this  organism 
were  very  great,  and  the  ingenuity  of  Nocard  and  Roux 2  was  required 
for  the  demonstration  of  its  existence.  Very  exactiiig  in  its  choice  of 
nutritive  material,  it  was  first  cultivated  in  the  fluids  of  the  rabbit, 
a  species  endowed  with  an  absolute  immunity  against  bovine  pleuro- 

1  Ztschr.f.  Hyp.,  Leipzig,  1893,  Bd.  xin,  S.  357. 
a  Ann.  de  Vlnst.  Pasteur,  Paris,  1898,  t.  xn,  p.  240. 


Immunity  against  pathogenic  micro-organisms    131 

pneumonia.     It  is  unnecessary  to  multiply  examples  to  obtain  a 
general  proof  that  natural  immunity  against  micro-organisms  cannot 
be  explained  by  the  incapacity  of  these  pathogenic  agents  to  live  in  [139] 
the  fluids  of  the  refractory  organism. 

We  must,  however,  ascertain  what  takes  place  in  resistant  animals 
inoculated  with  micro-organisms.  Here,  again,  it  is  preferable  to 
begin  with  the  lower  animals  of  simple  organisation.  We  have  already 
seen  that  examples  of  natural  immunity  are  not  rare  in  the  Inver- 
tebrata.  When  engaged  in  the  study  of  the  disease  found  in 
Daphniae,  small  Crustacea  so  common  in  fresh  water,  I  was  able 
to  show  that  the  special  Blastomycetes  which  cause  it  meet  with 
a  vigorous  resistance  on  the  part  of  the  organism.  As  the  Daphniae 
are  small,  transparent,  and  consequently  easily  observed  under  the 
microscope,  I  was  able  without  difficulty  to  establish  the  main 
phenomena  observable  in  these  organisms.  I  can  be  the  more  brief 
in  describing  these  phenomena  of  resistance  as,  in  addition  to  de- 
voting a  special  memoir  to  the  Daphnia  disease1,  I  have,  in  my 
Lectures  on  Inflammation  (pp.  97 — 103)2,  described  at  some  length 
the  reaction  of  their  organism  to  the  Monospora.  It  is  nevertheless 
necessary  that  I  should  recall,  very  briefly,  the  mechanism  by  which 
these  small  crustaceans  secure  immunity. 

The  spores  of  the  parasite — very  delicate  and  rigid  needles — are 
swallowed  with  the  food.  By  means  of  their  sharp  points  they 
perforate  the  intestine  and  penetrate  into  the  body  cavity,  full  of 
blood,  where  they  find  themselves  exposed  to  the  attacks  of  leuco- 
cytes. These  leucocytes,  guided  by  their  tactile  sense,  gather  around 
the  foreign  body,  ingest  it  completely  and  destroy  it.  It  is  remark- 
able that  the  spore,  which  is  furnished  with  a  very  resistant 
membrane,  once  in  the  interior  of  the  mass  of  leucocytes,  undergoes 
modifications  which  afford  evidence  of  the  presence  in  these  cells 
of  an  extraordinary  digestive  power.  The  surface  of  the  spore,  from 
being  smooth  and  regular,  becomes  pitted  and  sinuous,  the  spore 
breaks  up  into  fragments  and  is  reduced  to  a  mass  of  debris  which, 
in  the  form  of  brown  granules,  remains  indefinitely  in  the  contents 
of  the  leucocytes.  From  this  it  is  evident  that  these  phagocytes 
must  produce  a  ferment  which  is  capable  of  digesting  the  cellulose 
or  analogous  substance  which  forms  the  membrane  of  the  spore. 
Unfortunately,  the  small  size  of  the  Daphniae,  so  useful  for  the 

1  Virchow's  Archiv,  Berlin,  1884,  Bd.  xcvi,  S.  177. 

2  [English  translation,  pp.  83—86.] 

9—2 


132  Chapter  VI 

direct  observation  of  the  phenomena  of  immunity,  presents  an  insur- 
mountable obstacle  to  the  study  of  its  leucocyte  ferments,  especially 
in  vitro. 

[HO]  The  destruction  of  the  spores  of  the  parasite  by  the  leucocytes 
secures  to  the  Daphnia  a  real  immunity.  Of  a  hundred  Daphniae 
taken  in  my  aquarium  and  carefully  examined  under  the  microscope, 
fourteen  only  were  found  to  be  infected  by  the  budding  conidia  of 
the  parasite,  whilst  fifty-nine  of  the  others  contained  the  remains  of 
spores  that  had  been  destroyed  by  the  phagocytes.  When  transferred 
to  pure  water  containing  no  new  source  of  contagion,  these  Daphniae 
flourished  and  lived  a  normal  life,  giving  birth  to  a  numerous  progeny. 

The  immunity  of  the  Daphnia,  due  to  the  intervention  of 
phagocytes,  is  an  example  of  natural,  individual  immunity.  It  is 
not  the  specific  or  racial  possession  of  these  Crustacea,  for  when 
the  leucocytes  do  not  seize  the  spore,  at  once,  on  its  penetration  into 
the  body  cavity,  it  commences  to  germinate  and  gives  rise  to  a  whole 
generation  of  budding  cells.  These  cells,  then,  secrete  a  poison  which 
not  only  repels  the  leucocytes,  but  kills  and  completely  dissolves 
them.  Under  these  conditions  the  Daphnia  is  disarmed ;  the 
parasites  grow  in  the  organism,  deprived  of  its  arm  of  defence,  as 
in  a  culture  tube,  and  the  animal  rapidly  succumbs. 

Since  I  first  observed  this  struggle  between  the  Daphnia  and  its 
parasite,  some  eighteen  years  ago,  no  other  example  has  been  found 
that  is  so  easily  observed  and  so  demonstrative  of  the  protective 
action  of  phagocytes  in  an  animal  that  can  be  kept  under  observation, 
alive,  under  the  microscope.  Cases,  however,  are  not  wanting  in  the 
Invertebrata  in  which  the  different  phases  of  this  struggle  may  be 
observed  with  sufficient  accuracy  to  warrant  the  conclusion  that  in 
these  cases  also  the  phenomena  are  analogous  to  those  observed  in 
the  case  of  the  Daphniae. 

It  has  already  been  stated  in  Chapter  in.  that  the  larvae  of  the 
rhinoceros  beetle  (Oryctes  nasicornis),  although  very  sensitive  to  the 
cholera  vibrio,  are  very  refractory  to  anthrax  and  diphtheria.  In 
order  that  we  may  obtain  some  idea  of  the  mechanism  of  this  im- 
munity let  us  inject  into  the  body  cavity  of  these  large  white  grubs 
a  trace  of  anthrax  culture.  In  the  blood,  drawn  off*  the  following 
morning,  the  injected  bacilli  are  found,  not  in  the  plasma,  but  inside 
many  of  the  leucocytes.  Here  there  has  occurred,  as  in  the  Daphnia, 
an  ingestion  of  the  parasites  which  have  then  been  destroyed  by  the 
intracellular  digestion  of  phagocytes.  The  process  is  the  same,  then, 


Immunity  against  pathogenic  micro-organisms    133 

as  that  by  which  the  resorption  of  the  red  corpuscles  of  the  goose 
takes  place  when  they  are  injected  into  the  blood  of  cockchafer  larvae.  [141] 
In  both  cases  the  foreign  bodies  are  ingested  and  destroyed  by  the 
leucocytes  of  the  blood ;  this  act  of  resorption,  however,  taking  a  very 
long  time. 

Although  the  leucocytes  of  the  larvae  of  the  rhinoceros  beetle 
exhibit  a  positive  chemiotaxis  for  the  bacillus,  these  same  cells 
behave  in  a  very  different  fashion  in  presence  of  the  cholera  vibrio. 
Very  small  quantities  of  this  vibrio,  when  injected  into  the  blood  of  the 
larvae,  give  them  a  fatal  disease  :  the  vibrios  excite  in  the  leucocytes 
a  negative  chemiotaxis  and  flourish  without  hindrance  in  the  blood 
plasma.  The  larva  is  soon  transformed  into  a  culture  vessel  and  the 
numerous  vibrios  that  develop  in  it  cause  the  death  of  the  animal. 

The  difference  in  action  of  the  two  bacteria  cannot  be  explained 
by  any  corresponding  difference  in  their  mode  of  life  in  the  blood. 
Removed  from  the  organism  the  blood  plasma  of  the  white  larvae  of 
the  rhinoceros  beetle  is  a  culture  medium  just  as  favourable  to  the 
growth  of  the  anthrax  bacillus  as  to  that  of  the  cholera  vibrio. 
Moreover,  the  former  of  these  micro-organisms  is  quite  capable  of 
setting  up  a  fatal  disease  in  other  representatives  of  the  class  of 
Insects.  Kovalevsky1  has  discovered  in  the  house  cricket  four  phago- 
cytic  organs,  with  a  great  appetite  for  all  kinds  of  foreign  particles 
that  may  penetrate  into  its  body.  The  blood  of  mammals,  when 
injected  below  the  skin  of  the  cricket,  is  rapidly  absorbed  by  the 
cells  of  the  four  "spleens"  (for  so  Kovalevsky  designates  these 
phagocytic  organs).  The  resorption  of  the  red  blood  corpuscles  goes 
on  within  these  phagocytes  owing  to  their  power  of  intracellular 
digestion.  When  Kovalevsky  kept  crickets  at  a  temperature  of 
22°— 23°  C.  and  injected  them  with  anthrax  bacilli  he  noted  that 
these  bacilli  also  were  ingested  by  the  cells  of  the  spleens.  There 
was,  thus,  no  manifestation  of  negative  chemiotaxis  of  these  elements 
towards  the  bacillus.  The  ingestion  of  the  bacilli  by  the  phagocytes 
was  not  sufficient,  however,  to  protect  the  animal.  The  bacilli  re- 
produced themselves  rapidly  in  the  blood  fluid ;  the  intracellular 
lacunae  of  the  spleens  were  full  of  them  and  the  crickets  quickly 
succumbed  to  the  infection. 

Nevertheless  these  crickets  are  quite  capable  of  resisting  certain 
other  bacteria.  Balbiani2  has  shown  that  they  are  refractory  to  [112] 

1  Bull.  Acad.  d.  sc.  de  St  Peter sb.,  1894,  t.  xin,  p.  437. 

2  Compt.  rend.  Acad.  d.  sc.,  Paris,  1886,  t  cm,  p.  952. 


134  Chapter  VI 

a  great  number  of  bacilli  belonging  to  the  group  of  Bacillus  subtilis. 
He  observed  that  when  injected  into  the  body  of  the  cricket  these 
bacilli  are  devoured  and  destroyed  by  the  leucocytes  of  the  blood 
and  by  the  large  cells  of  the  pericardial  tissue  corresponding  to  the 
elements  of  the  spleens  of  Kovalevsky.  Whilst  the  crickets  and  other 
Orthoptera,  which  are  rich  in  phagocytes,  exhibit  a  real  immunity 
against  these  bacilli,  insects  which  have  very  few  leucocytes  such 
as  butterflies,  flies  and  Hymenoptera  are  found  to  be  much  more 
susceptible  to  infection  by  the  same  bacilli.  In  this  case  the  direct 
relation  between  immunity  and  phagocytosis  is  very  marked. 

The  Mollusca  also  furnish  some  interesting  examples  of  natural 
immunity.  Karlinsky1  has  observed  that  anthrax  bacilli,  when  in- 
jected into  the  blood  of  slugs  and  snails,  soon  disappear  from  their 
bodies ;  these  pulmonate  Gasteropods  are  absolutely  unaffected  by 
this  bacillus  so  formidable  for  many  species  of  animals.  From  the 
rapidity  of  this  disappearance  of  the  bacilli  it  has  even  been  con- 
cluded that  it  was  impossible  for  this  bacillus  to  live  in  the  fluids 
of  Mollusca.  Kovalevsky  (I.e.  p.  443)  has  studied  this  question  with 
the  carefulness  that  characterises  all  his  work.  He  confirms  the 
fact  that  snails  (Helix  pomatia)  resist  the  introduction  of  a  large 
quantity  of  anthrax  bacilli  into  their  bodies  ;  he  notes  also  that 
these  bacteria  disappear  from  the  blood.  But  he  finds  them  again 
in  the  tissues  of  the  foot,  and  especially  in  the  cells  which  surround 
the  pulmonary  vessels.  "The  greater  number  of  the  bacteria  are 
found  in  the  cells  of  that  part  of  the  pulmonary  region  in  Helix 
which  adjoins  the  heart  and  kidney.  All  the  bacteria  were  ingested 
by  the  cells  and  I  easily  succeeded  in  demonstrating  this  not  only  in 
sections  but  also  in  bulk"  (p.  444).  The  snails  remained  in  good 
health  in  spite  of  the  presence  in  their  phagocytes  of  numerous 
bacteria  which  maintained  themselves  there  for  some  time.  At  the 
end  of  ten  or  twelve  days  and  more  these  bacteria  still  presented 
their  usual  aspect ;  this  accords  well  with  the  slowness  with  which 
intracellular  digestion  goes  on  in  the  majority  of  the  Irivertebrata. 
These  bacteria  were,  however,  no  longer  living,  although  still  un- 
[143]  digested.  Morsels  of  the  pulmonary  tissue  of  the  snails  that  were 
injected  with  anthrax  bacilli  still  gave  cultures  48  hours  after  in- 
jection and  contained  bacilli  capable  of  giving  fatal  anthrax  to  mice. 
Later,  media  seeded  with  similar  particles  remained  sterile,  and  mice 
inoculated  therewith  continued  to  live.  From  these  experiments  it  may 
1  Centralbl.f.  Bakteriol.  u.  Parasitenk.,  Jena,  1889,  Bd.  v,  S.  5. 


Immunity  against  pathogenic  micro-organisms    135 

be  accepted  that  bacteria,  living  in  the  blood  plasma,  become  the 
prey  of  phagocytes  which  render  them  inoffensive  and  kill  them. 
This  example  demonstrates  once  again  that  the  organism  gets  rid  of 
bacteria  by  the  same  mechanism  as  that  which  serves  for  the  re- 
sorption  of  any  of  the  formed  elements.  The  snail  reacts  to  bacteria 
as  it  does  to  the  red  corpuscles  of  the  goose. 

It  is  unnecessary  to  insist  further  on  the  natural  immunity  of  the 
Invertebrata,  and  it  is  useless  to  multiply  examples  which  always 
point  in  the  same  direction:  to  the  importance  of  phagocytic  reaction 
and  of  intracellular  digestion  in  resorption  and  immunity.  We  must 
pass  on  to  the  examination  of  the  reaction  phenomena  of  the 
vertebrate  organism  towards  pathogenic  micro-organisms,  following, 
as  hitherto,  the  comparative  method.  We  will  commence  with  the 
study  of  the  natural  immunity  of  fishes  as  lower  representatives  of 
the  great  group  of  the  Vertebrata. 

It  is  well  known  that  fishes  are  liable  to  infective  diseases  and 
pisciculture  has  often  to  deplore  considerable  losses  brought  about 
sometimes  by  certain  of  the  lower  Fungi  (e.g.  Saprolegniae),  some- 
times by  Bacteria.  The  pathogenic  microbes  which  produce  epi- 
demics in  fishes  are  still  little  understood;  but  among  the  bacteria 
•which  kill  many  of  the  higher  animals  are  some  which  cause  fatal 
maladies  in  certain  fishes.  Thus  the  anthrax  bacillus  so  virulent  for 
many  mammals  is  capable  also,  as  we  have  seen,  of  producing  an 
infection  in  the  cricket,  and  may  cause  the  death  of  small  marine 
osseous  fishes,  the  Hippocampi.  Sabrazes  and  Colombot1,  who  have 
studied  this  question,  have  demonstrated  that  the  anthrax  bacillus, 
which  is  virulent  for  the  rabbit,  when  inoculated  into  these  fishes 
first  produces  swellings  at  the  seat  of  inoculation  and  ultimately 
becomes  generalised  throughout  the  body,  producing  a  fatal  septi- 
caemia. As  these  experiments  have  given  this  result  at  a  temperature 
of  14° — 16°  C.,  it  is  quite  evident  that  the  bacillus,  in  order  to 
manifest  its  pathogenic  effect,  in  no  way  needs  the  high  temperature  [144] 
of  the  mammalian  body  for  its  action. 

Now  among  fishes  there  are  not  wanting  species  which  resist  the 
anthrax  bacillus.  Mesnil2  has,  in  our  laboratory,  thoroughly  studied 
the  mechanism  of  this  immunity.  He  has  shown  that  several  fresh- 
water fishes,  e.g.  the  perch  (Perca  fluviatilis),  the  gudgeon  (Gobio 
fluviatilis),  and  the  gold-fish  (Carassius  auratus),  will  resist  an 

1  Ann.  de  tlnst.  Pasteur,  Paris,  1894,  t.  vin,  p.  696. 
3  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  301. 


136  Chapter  VI 

injection  of  a  considerable  number  of  bacilli  into  the  abdomen. 
When  kept  at  temperatures  of  15°— 20°  C.  or  even  23°  C.,  a  tem- 
perature at  which  the  bacilli  are  able  to  develop  very  abundantly, 
these  fishes  destroy  a  large  number  of  the  bacteria  in  their  bodies. 
Soon  after  the  introduction  of  the  bacilli  into  the  peritoneal  cavity,  the 
numerous  leucocytes  accumulate  around  them  and  ingest  them  by  the 
same  mechanism  that  is  observed  in  the  Invertebrata  or  in  the  same 
fishes  when  absorbing  the  red  blood  corpuscles  of  alien  species.  In 
the  gudgeon,  at  as  early  as  six  and  a  half  hours,  a  very  marked,  nay, 
an  almost  complete  phagocytosis  is  set  up. 

It  is  impossible  to  doubt  the  fundamental  fact  that  the  bacilli, 
at  the  moment  of  their  ingestion,  are  in  a  perfect  condition  of 
vitality  and  virulence.  The  fluid  of  the  peritoneal  exudation,  when 
withdrawn  from  the  animal,  is  of  itself  incapable  of  preventing  the 
development  of  the  anthrax  bacilli.  The  peritoneal  lymph  of  the 
above-mentioned  fishes  is,  in  vitro,  even  a  good  culture  medium  for 
these  bacilli. 

When,  long  after  the  completion  of  the  phagocytosis  by  the 
leucocytes  of  the  peritoneal  exudation,  a  drop  of  the  exudation  is 
withdrawn  and  kept  outside  the  organism  under  suitable  conditions 
of  temperature  and  moisture,  a  number  of  the  ingested  bacilli  begin  to 
multiply  and  give  an  abundant  culture.  This  experiment  proves,  in- 
disputably, that  the  bacilli  are  devoured  in  the  living  state.  If  a  little 
of  the  peritoneal  exudation,  withdrawn  several  (up  to  nine)  days  after 
the  injection  of  the  bacilli,  be  injected  below  the  skin  of  guinea-pigs 
these  animals  die  from  generalised  anthrax,  a  fact  which  demonstrates 
that  the  bacilli,  which  have  been  ingested  alive,  have  retained  their 
virulence  a  long  time  after  they  have  been  devoured  by  the  leuco- 
cytes. But,  if  the  peritoneal  exudations  that  have  been  withdrawn  at 
still  longer  periods  after  injection  be  examined,  it  is  found  that  they 
[145]  no  longer  contain  bacilli  capable  of  developing  in  culture  media  or 
of  setting  up  the  disease  in  the  most  susceptible  animal.  Hence  it 
follows  that  in  the  organism  of  the  refractory  fish,  the  bacteria  are 
not  destroyed  by  the  fluids  but  by  the  phagocytes,  which  take  a  long 
time  to  bring  about  the  complete  intracellular  digestion  of  ingested 
micro-organisms. 

The  phagocytes  which  assure  immunity  to  the  osseous  fishes  that 
were  studied  by  Mesnil  belong  principally  to  the  group  of  haemo- 
macrophages.  These  are  leucocytes  with  abundant  protoplasm 
which  stain  readily  by  basic  aniline  dyes,  rnononuelear  cells  whose 


Immunity  against  pathogenic  micro-organisms    137 

nucleus,  however,  is  sometimes  divided  into  lobes.  It  is  to  be  noted 
that  in  the  perch  these  are  the  sole  representatives  of  the  motile 
phagocytes,  and  that  in  this  fish  not  only  the  eosinophile  but  every 
other  variety  of  granular  leucocyte  is  completely  wanting.  In  the 
gudgeon,  in  addition  to  haemomacrophages,  some  microphages  whose 
protoplasm  stains  faintly  with  acid  aniline  colours  are  met  with. 
These  facts  will  be  useful  to  us  when  we  come  to  study  the  part 
played  by  phagocytes  in  immunity  from  a  general  point  of  view. 

Another  class  of  cold-blooded  animal,  the  Amphibia,  has  been 
much  more  frequently  studied  from  the  point  of  view  of  infection 
and  immunity.  The  frog,  an  animal  so  convenient  for  many  physio- 
logical and  pathological  researches,  has  been  much  employed  for 
the  study  of  immunity  against  pathogenic  micro-organisms.  Quite 
a  literature,  which  has  been  excellently  summarised  in  the  memoir  of 
Mesnil  already  cited,  and  to  which  we  shall  have  occasion  to  return 
more  than  once,  has  been  accumulated  on  the  subject. 

The  immunity  of  frogs  against  the  anthrax  bacillus  was  early 
demonstrated  and  studied  in  Robert  Koch's  celebrated  memoir1 
on  anthrax.  This  observer,  after  injecting  an  emulsion  of  anthrax 
spleen  into  the  lymph  sac  of  the  frog,  recovered  the  bacilli  from 
the  interior  of  round  cells  which  burst  readily  when  transported 
into  water.  Koch,  accepting  the  view  then  generally  held,  thought 
that  the  bacilli  found  a  favourable  culture  medium  in  the  contents 
of  certain  cells,  but  that,  in  spite  of  this,  the  frog  was  capable  of 
manifesting  a  real  immunity  against  anthrax.  Gibier2  made  the  [146] 
interesting  discovery  that  frogs  when  subjected  to  the  influence 
of  high  temperature  (about  37°  C.)  lose  their  natural  immunity  and 
readily  contract  fatal  anthrax. 

Since  that  time  the  mechanism  by  which  the  organism  of  the  frog 
secures  immunity  against  the  anthrax  bacillus  has  repeatedly  been 
studied.  In  a  memoir  which  appeared  in  18843  I  insisted  that  the 
principal  part  played  in  this  immunity  belonged  to  the  phagocytes 
which  devour  the  injected  bacteria  and  subject  them  to  intra- 
cellular  digestion.  The  round  cells  described  by  Koch  are  nothing 
but  the  leucocytes  of  the  lymph  sac  which  have  seized  upon  the 
anthrax  bacilli.  These  bacilli  instead  of  thriving  in  the  cell  contents 
find  there  a  very  unfavourable  medium,  and  perish  at  the  end  of 

1  Cohn's  "  Beitrage  zur  Biologie  der  Pflanzen,"  Breslau,  1876,  Bd.  n,  S.  300. 

2  Compt.  rend.  Acad.  d.  sc.,  Paris,  1882,  t.  xciv,  p.  1605. 

3  Virchows  Archie,  Berlin,  1SS4,  Bd.  xcvn,  S.  502. 


138  Chapter  VI 

a  longer  or  shorter  period.  When  the  activity  of  the  phagocytes  is 
impeded  by  unfavourable  influences,  e.g.  high  temperature,  they 
exhibit  a  very  feeble  reaction,  incapable  of  assuring  to  the  frog  that 
immunity  which,  under  normal  conditions,  it  possesses.  The  con- 
clusions I  have  just  summarised  have  raised  very  lively  opposition 
from  a  large  number  of  observers.  Baumgarten1,  with  his  pupils 
Petruschky2  and  Fahrenholtz3,  have  endeavoured  to  demonstrate  that 
phagocytosis  plays  no  part  in  immunity  and  that  the  frogs  resist 
anthrax  simply  because  the  bacilli  are  incapable  of  maintaining 
themselves  alive  in  the  fluids  of  this  Batrachian.  Nuttall4,  of 
Fliigge's  school,  also  maintained  that  frogs  resist  anthrax  owing  to 
the  bactericidal  power  of  their  fluids.  This  view  has  been  defended 
by  several  other  observers  and  appeared  for  some  time  to  become 
quite  dominant. 

Nevertheless,  it  is  possible  to  demonstrate  that  the  plasmas  of 
the  frog  not  only  are  not  inimical  to  the  life  of  the  bacillus,  but 
serve  as  a  good  culture  medium  for  it5.  All  that  is  necessary  for  the 
demonstration  of  this  fact  is  to  introduce  below  the  skin  of  frogs 
[147]  anthrax  spores  enclosed  in  a  sac  of  reed  pith,  or  simply  enveloped  in 
a  small  piece  of  filter-paper.  The  plasma  of  the  lymph  sac  at  once 
permeates  the  spores  and  allows  them  to  germinate  and  produce 
quite  a  generation  of  bacilli.  But,  as  soon  as  the  leucocytes  pass 
through  the  paper,  they  seize  upon  the  young  bacilli,  digest  them 
in  their  substance  and  prevent  their  pathogenic  action.  The  germi- 
nation of  the  spores  may  take  place  even  where  they  have  been 
introduced  below  the  frog's  skin  without  being  protected  in  any  way 
whatever.  But,  under  these  conditions,  only  a  certain  number  of  the 
spores  germinate,  the  majority  not  having  time  to  do  so  before  the 
arrival  of  the  leucocytes.  The  small,  very  short  bacilli  which  proceed 
from  the  germinated  spores,  are,  along  with  the  spores  that  have 
not  germinated,  soon  ingested  by  the  phagocytes.  But,  whilst  the 
rods  are  in  the  end  digested  within  these  cells,  the  ingested  spores 
remain  intact  for  a  very  long  time  :  they  do  not  germinate,  but  they 
are  not  destroyed  and  retain  their  vitality  indefinitely,  in  spite  of 

1  Centralblf.  klin.  Med.,  Bonn,  1888,  S.  516. 

2  "  Untersuch.  iiber  d.  Immunitat  d.  Frosches  gegen  Milzbrand,"  Ziegler's  Beitr. 
z.path.  Anat.,  Jena,  1888,  Bd.  in,  S.  357. 

3  "Beitrage  z.  Kritik  der  Metschnikoff'schen   Phagocytenlehre,"  Inaug.   Diss., 
Konigsberg,  1889. 

4  Ztschr.f.  Hyg.,  Leipzig,  1888,  Bd.  iv,  S.  353. 

8   Virchouts  Archiv,  Berlin,  1888,  Bd.  cxiv,  8.  466. 


Immunity  against  pathogenic  micro-organisms     139 

the  influence  of  the  phagocytes.  It  is  sufficient  to  withdraw  from 
a  frog,  that  has  been  inoculated  with  anthrax  spores  some  time 
before  and  kept  at  a  moderate  temperature  (15° — 25°  C.),  a  little 
lymph  and  sow  it  in  any  nutrient  medium  (of  those  employed  in 
the  culture  of  bacteria),  in  order  to  see  the  spores  germinate  and 
produce  a  whole  generation  of  absolutely  normal  filamentous  bacilli. 
All  these  phenomena  have  been  carefully  studied  by  Trapeznikoff1 
in  a  work  executed  in  my  laboratory.  It  is  obvious  from  his  experi- 
ments that  the  phagocytes  of  the  frog  are  quite  capable  of  protecting 
the  organism  against  the  anthrax  bacillus  by  ingesting  and  digesting 
the  bacilli  in  the  vegetative  state  and  by  preventing  the  germination 
of  the  ingested  spores.  This  phagocytic  action  is  very  important  in 
presence  of  the  fact  that  the  plasmas  of  the  frog  allow  the  spores  to 
germinate  and  the  bacilli  to  develop  and  produce  abundant  cultures. 

The  immunity  of  frogs  against  the  anthrax  bacillus  that  we  have 
just  described  and  which  is  guaranteed  by  the  activity  of  the 
phagocytes,  is  constant  under  the  conditions  of  temperature  above 
mentioned  (15° — 25°  C.),  conditions  which  are  sufficient,  however, 
to  ensure  the  death  of  susceptible  cold-blooded  animals,  such  as 
the  cricket  or  Hippocampus,  from  anthrax.  The  edible  frog,  a 
species  that  readily  accommodates  itself  to  a  temperature  of  35°  C.,  [148] 
resists,  even  under  these  conditions,  infection  by  the  bacillus,  as 
pointed  out  by  Mesnil  in  a  work  already  cited  when  treating  of  the 
immunity  of  fishes.  The  green  frog  (Eana  esculenta)  when  kept  for 
a  long  time  at  this  high  temperature,  so  suitable  for  the  development 
of  the  anthrax  bacillus,  reacts  by  the  same  phagocytic  mechanism. 
The  leucocytes  of  the  lymph  and  blood,  the  cells  of  the  splenic  pulp 
and  Kupffer's  stellate  cells  of  the  liver,  seize  the  introduced  bacilli 
and  digest  them  as  in  any  other  case  of  phagocytosis.  The  brown 
frog  (Eana  temporaria)  adapts  itself  but  slightly  and  with  great 
difficulty  to  the  high  temperature  and  dies  whether  it  has  been 
inoculated  with  anthrax  or  not.  Under  these  conditions  the  bacteria 
develop  in  the  body  of  the  dead  or  dying  frogs,  but  Mesnil  insists 
on  the  fact  that  a  true  anthrax  infection  is  not  produced,  as  has 
been  maintained  by  Gibier  as  the  outcome  of  his  researches. 

Dieudonne2,  however,  has  found  a  method  of  removing  the 
natural  immunity  of  the  frog  against  the  anthrax  bacillus,  by  inocu- 
lating it  with  an  artificial  bacterial  race  which  he  had  adapted  to 

1  Ann.  de  FInst.  Pasteur,  Paris,  1891,  t.  v,  p.  362. 

2  Arb.  a.  d.  k.  Gsndhtsamte,  Berlin,  1894,  Bd.  ix,  S.  497. 


140  Chapter  VI 

develop  fairly  luxuriantly  at  the  low  temperature  of  12°C.  Under 
these  conditions  all  the  inoculated  frogs,  even  those  which  had 
resisted  the  inoculation  with  ordinary  bacteria  (grown  at  37*5°  C.), 
died  within  a  period  of  48  to  56  hours,  containing  many  bacilli  in 
the  blood  and  organs.  Dieudonn^  has  not  studied  the  essential 
mechanism  that  accompanies  this  loss  of  immunity;  but  it  is  very 
probable  that,  for  one  thing,  we  have  here  to  do  with  a  reinforcement, 
special  for  the  frog,  of  the  bacillus  that  has  become  accustomed 
to  develop  at  a  low  temperature.  This  bacillus  must  multiply,  in 
frogs  that  have  been  maintained  at  a  low  temperature,  much  more 
rapidly  and  profusely  than  would  the  ordinary  bacillus.  On  the  other 
hand,  the  susceptibility  of  Dieudonn^'s  frogs  must  depend  on  a  less 
resistance  of  the  organism  under  the  conditions  of  his  experiments. 
Unfortunately,  we  cannot  find  in  his  memoir  sufficient  data  on  these 
points;  he  does  not  even  state  the  temperature  at  which  the  frogs 
that  had  been  inoculated  with  bacteria  adapted  to  cold  lived. 
Dieudonne  invokes  the  analogy  of  his  results  with  those  obtained 
[149]  in  the  case  of  the  immunity  and  susceptibility  of  frogs  as  regards 
a  septicaemic  bacillus. 

This  bacillus  (Bacillus  ranicida)  has  been  made  the  subject  of 
an  interesting  study  by  Ernst1.  It  is  a  small,  very  slender  bacillus, 
which,  in  frogs,  produces  a  fatal  malady  epidemic  in  spring,  but 
ceasing  completely  during  summer.  Taking  this  fact  as  a  basis, 
Ernst  has  succeeded  in  conferring  immunity  upon  frogs  in  autumn 
by  placing  them  in  an  incubator  at  25°  C.  In  spite  of  the  injection 
of  a  considerable  dose  of  the  small  bacillus,  the  frogs  living  at  this 
temperature  remained  in  good  health,  whilst  control  animals  exposed 
to  a  low  temperature  died  of  septicaemia.  The  counter-test  was 
made  in  summer.  Inoculated  frogs  that  were  kept  in  the  laboratory 
were  unaffected,  whilst  those  that  had  been  kept  in  a  refrigerating 
apparatus  at  6° — 10°  C.  invariably  died.  It  may  be  asked,  Is  this 
evident  influence  of  temperature  on  immunity  and  receptivity  exer- 
cised on  the  organism  of  the  frog  or  upon  the  pathogenic  bacillus  ? 
In  the  case  where  a  bacillus  can  only  develop  at  low  temperatures 
its  harmlessness  at  the  higher  temperature  may  be  readily  under- 
stood. The  experiments  of  Ernst  have  demonstrated,  however,  that 
this  small  bacillus  develops  much  better  at  22°  C.,  and  even  at  30°  C., 
than  at  lower  temperatures.  It  must  be  concluded,  therefore, 
that  the  high  temperature  which  confers  immunity  acts  not  by 
1  Ziegler's  Beitr.  z.path.  Anat.,  Jena,  1890,  Bd.  vm,  S.  203. 


Immunity  against  pathogenic  micro-organisms    141 

weakening  the  bacillus,  but  rather  by  reinforcing  the  resisting  power 
of  the  organism.  The  low  temperatures  (6° — 10°  C.)  that  are  favour- 
able to  a  fatal  infection  have  a  different  action;  that  is  to  say, 
they  weaken  the  reaction  of  the  inoculated  frogs. 

Although  Ernst  has  not  studied  the  mechanism  of  this  resistance 
fully,  it  is  evident,  from  the  data  he  has  supplied,  that  it  consists  in 
a  phagocytic  reaction.  He  was  able  to  demonstrate  the  ingestion  of 
the  bacilli  by  the  phagocytes  in  the  susceptible  refrigerated  frogs,  as 
well  as  in  the  refractory  frogs,  kept  at  a  higher  temperature;  but  in 
the  former  case  the  phagocytosis  was  so  feeble  that  24  hours  after 
inoculation  a  considerable  number  of  free  bacilli  were  still  found 
in  the  lymph  of  the  dorsal  sac,  whilst  in  the  refractory  frogs  the 
much  more  active  phagocytosis  brought  about  the  disappearance  of 
the  free  bacilli  during  the  first  day.  If,  as  is  very  probable,,  the 
analogy  of  this  septicaemia  with  anthrax  in  frogs,  upon  which  Ernst 
insists,  really  exists,  it  must  be  concluded  that  the  susceptibility  of  [150] 
these  Batrachians  to  the  modified  race  of  the  bacillus  depends  on 
their  weak  phagocytic  resistance. 

Since,  in  these  two  examples  of  natural  immunity  in  the  frog,  we 
have  seen  that  the  phagocytic  activity  exhibits  itself  in  an  active 
form  against  bacteria  which  readily  develop  in  the  fluids  of  the 
same  animal,  we  might  conclude  that  the  reaction  of  the  phagocytes 
constitutes  a  general  mode  of  defence  in  cold-blooded  animals. 
But  Lubarsch1,  a  very  cautious  observer,  has  expressed  an  opposite 
view,  based  on  his  studies  on  the  bacillus  of  mouse  septicaemia. 
He  convinced  himself  that  frogs  will  resist  injections  of  even  con- 
siderable quantities  of  this  bacillus,  without  any  co-operation  on 
the  part  of  the  phagocytes.  As  we  have,  here,  to  do  with  a 
matter  of  fact,  Mesnil  (Lc.)  set  himself  to  verify  these  observations, 
with  the  object  of  establishing  whether  it  was  a  case  of  a  real 
exception  or  of  a  simple  misunderstanding.  He  was  able  to  de- 
monstrate, by  irrefutable  observations  and  experiments,  that  the 
bacilli  of  mouse  septicaemia  when  inoculated  into  frogs,  set  up  a 
very  pronounced  positive  chemiotaxis  on  the  part  of  the  phagocytes, 
which  seized  and  digested  the  bacilli  just  as  they  do  the  anthrax 
bacillus.  This  apparent  exception,  therefore,  becomes  transformed 

1  Centralbl.f.  Bakteriol.  u.  Parasitenk.,  Jena,  1889,  Bd.  vi,  SS.  481  and  529; 
Fortschr.  d.  Med.,  Berlin,  1890,  Bd.  vm,  S.  665 ;  Ztschr.  f.  klin.  Med.,  Berlin,  1891 ; 
"  Ueber  Immunitat  u.  Schutzimpfung,"  Schneidemiihrs  Thiermed.  Vortrage,  1892, 
Bd.  n. 


142  Chapter  VI 

into  an  additional  argument  in  favour  of  phagocytic  reaction  being 
a  general  factor  in  immunity.  In  support  of  this  hypothesis  I  may 
adduce  a  further  example,  already  mentioned  in  a  preceding  chapter 
when  discussing  another  question.  The  frog  is  very  refractory  against 
the  cholera  vibrio.  When  these  vibrios  are  inoculated  into  the  dorsal 
lymphatic  sac  or  into  any  other  part  of  the  body  the  animal  retains 
its  health  unimpaired.  An  examination  of  the  exudation  at  the  point 
of  inoculation  demonstrates  that  the  vibrios  meet  with  a  vigorous 
opposition  on  the  part  of  the  phagocytes,  which  ingest  and  com- 
pletely digest  them.  This  is  of  special  interest  from  the  fact  that 
the  frog  is  very  sensitive  to  the  toxin  of  the  cholera  vibrio.  When 
injected  in  a  weak  dose  it  kills  the  frog  very  quickly.  Two  small 
frogs  died  in  less  than  an  hour  from  the  effect  of  0'5  c.c.  of  cholera 
toxin. 

The  natural  immunity  of  the  frog  against  the  cholera  vibrio  affords, 
[151]  then,  an  example  in  which  the  organism,  destroying  the  vibrio  by 
phagocytosis,  prevents  the  production  of  the  poison,  which,  otherwise, 
would  infallibly  kill  it. 

Having  demonstrated  that  phagocytic  reaction  manifests  itself  in 
the  frog  in  all  cases  of  natural  immunity  that  have  been  sufficiently 
studied,  we  must  dwell  for  an  instant  on  the  question  of  the  con- 
dition of  the  bacteria  at  the  moment  of  their  ingestion  by  the 
phagocytes.  It  is  very  evident  that  this  phagocytic  defence  is  only 
efficient  on  condition  that  it  is  exercised  against  bacteria  which,  in 
its  absence,  might  injure  the  organism  by  their  multiplication  and 
their  virulence.  For  this  reason  the  question  as  to  whether  the 
micro-organisms,  before  being  ingested,  were  living  and  capable  of 
producing  their  pathogenic  action  has  been  widely  discussed.  It 
has  even  been  suggested  that  the  phagocytes  are  only  capable  of 
ingesting  the  dead  bodies  of  micro-organisms  that  have  been  killed 
by  other  agents.  Frogs  are  very  suitable  for  a  study  of  this  question. 
When  a  drop  of  the  exudation  is  removed  some  time  after  inocu- 
lation with  a  motile  organism,  such  as  the  Bacillus  pyocyaneus  or 
the  cholera  vibrio,  the  organism  was  often  found  moving  rapidly 
within  the  vacuoles  inside  leucocytes.  The  experiment  will  succeed 
even  more  completely  if  a  drop  of  frog's  lymph  be  mixed,  on  a  slide, 
with  a  trace  of  a  culture  of  these  motile  micro-organisms,  the  latter 
being  soon  found  in  the  clear  vacuoles  included  in  leucocytes  and 
executing  extremely  rapid  movements. 

Besides  this  direct  proof  we  can  assure  ourselves  of  the  living 


Immunity  against  pathogenic  micro-organisms    143 

condition  of  the  micro-organisms  in  another  way.  Withdraw  a  drop 
of  the  exudation  at  an  advanced  stage  of  the  process  when  there 
are  no  longer  any  free  micro-organisms ;  inside  the  phagocytes  a  few 
scattered  bacteria,  more  or  less  well  preserved,  can  still  be  seen.  It 
is  sufficient  to  keep  a  hanging  drop  of  such  an  exudation  at  a  tem- 
perature of  about  30°  C.,  care  being  taken  to  keep  it  from  drying,  but 
without  adding  to  it  any  nutrient  medium.  Under  these  conditions 
the  leucocytes  die  more  or  less  rapidly,  but  the  bacteria  regain  vigour: 
they  begin  to  multiply,  and  at  the  end  of  a  short  time  produce  a 
generation  of  bacteria  within  the  dead  leucocyte.  The  multiplication 
of  the  bacteria  goes  on  progressively  and  the  hanging  drop  is  trans- 
formed into  a  real  pure  culture.  Mesnil  was  able  to  confirm  these 
data  with  the  exudations  of  frogs  that  had  been  inoculated  with  either 
the  bacilli  of  anthrax  or  of  mouse  septicaemia. 

The  bacteria,  ingested  in  the  living  state  by  phagocytes,  retain  [152] 
their  original  virulence.  Some  authors  think,  and  I  was  formerly  of 
this  opinion,  that  at  the  end  of  a  more  or  less  prolonged  sojourn 
within  the  leucocytes,  anthrax  bacilli  undergo  an  attenuation  in 
their  virulence.  Later,  numerous  researches  have,  however,  de- 
monstrated that  this  opinion  is  incorrect,  and  that  the  virulence  is 
maintained  in  the  bacteria  included  in  the  phagocytes  of  frogs  the 
whole  time  that  these  bacteria  remain  alive.  Dieudonne*  has  in- 
sisted on  this  fact  as  regards  the  anthrax  bacillus.  Mesnil  has 
confirmed  it  for  this  same  species  and  for  the  bacillus  of  mouse 
septicaemia.  It  is  impossible,  therefore,  to  doubt  this  general  result, 
that  frogs  which  are  refractory  against  certain  bacteria  resist  because 
of  the  phagocytosis  which  is  exercised  against  living  and  virulent 
micro-organisms. 

We  have  insisted  sufficiently  on  the  analysis  of  the  natural 
immunity  of  the  frog,  and  need  not  tarry  over  the  facts  relating  to 
other  amphibia  which,  moreover,  have  been  much  less  studied.  The 
reptiles,  those  higher  representatives  of  the  Vertebrata  called  cold- 
blooded, often  present  examples  of  really  remarkable  immunity.  Thus 
alligators  will  resist  enormous  doses  of  various  bacteria,  such  as 
the  anthrax  bacillus,  that  of  human  tuberculosis  or  the  cocco-bacillus 
of  typhoid  fever.  When,  some  time  after  an  injection  is  made,  the 
exudation  at  the  point  of  inoculation  is  withdrawn  there  is  found 
a  large  number  of  leucocytes,  amongst  which  may  be  recognised 
many  eosinophile  microphages,  though  the  majority  are  macrophages 
with  one,  two  or  more  nuclei.  Really  giant  cells  are  found  in  the 


144  Chapter  VI 

exudation,  It  is  the  macrophages  which  specially  manifest  phago- 
cytosis and  they  are  often  found  crammed  with  the  injected  bacteria, 
as  I  was  able  to  assure  myself  after  injections  of  typhoid  cocco-bacilli. 
The  natural  immunity  of  alligators  (Alligator  mississipiensis)  persists 
not  only  at  the  temperature  of  the  incubator  (37°  C.),  but  also  at  room 
temperature  (20°— 22°  C.). 

Passing  in  review  the  animal  kingdom  we  must  pause  for  a 
moment  to  consider  the  natural  immunity  of  birds  or  lower  warm- 
blooded Vertebrates.  The  classic  example  of  this  immunity  is  that 
of  the  fowl  against  anthrax.  It  has  long  been  known  that  birds  resist 
[153]  inoculation  with  anthrax  or  only  exhibit  a  feeble  receptivity;  though 
smaller  birds  are  for  the  most  part  susceptible  to  anthrax,  the  pigeon 
is  much  less  so  and  the  fowl  presents  a  case  of  the  most  pronounced 
immunity.  It  was  believed  to  be  absolutely  refractory  until  the 
experiments  of  Pasteur  and  Joubert1,  who  found  a  sure  method  of 
suppressing  this  immunity.  Fowls  that  had  been  inoculated  with 
the  bacillus  were  immersed  up  to  the  thighs  in  cold  water  in  order  to 
bring  down  their  temperature.  It  was  found  that,  under  these  con- 
ditions, the  anthrax  bacillus  developed  at  the  seat  of  inoculation  and 
later  became  generalised  in  the  blood,  and  invariably  caused  death. 
It  was  concluded  from  this  that  the  natural  immunity  of  the  fowl 
was  dependent  on  its  very  high  normal  temperature  (41° — 42°)  which 
interfered  with  the  pathogenic  functions  of  the  anthrax  bacillus. 

Hess2  studied  the  mechanism  of  this  immunity  of  the  fowl  and 
pointed  out  the  important  part  that  phagocytosis  plays  in  the  de- 
struction of  the  inoculated  bacteria. 

These  researches  were  resumed  in  my  laboratory  by  Wagner3. 
Having  established  that  the  anthrax  bacillus  develops  readily  in  the 
blood  and  the  blood  serum  of  fowls,  outside  the  organism,  at  high 
temperatures  (42° — 43°  C.),  he  came  to  the  conclusion  that  the 
lowering  of  the  temperature  of  the  body  of  the  fowls  by  immersing 
them  in  water  produced,  not  a  reinforcement  of  the  bacillus,  but 
a  weakening  of  the  resisting  power  of  the  animal.  He  was  able 
to  convince  himself  that  this  resistance  exhibits  itself  in  the  activity 
of  the  phagocytes  which  ingest  and  destroy  the  anthrax  bacillus  in 
its  vegetative  state.  In  the  normal  fowl  the  phagocytosis  is  rapid 
and  very  pronounced,  whilst  in  a  fowl  that  has  been  refrigerated  this 

1  Bull  Acad.  de  med.,  Paris,  1878,  p.  440. 

2  Virchow's  Archiv,  Berlin,  1887,  Bd.  cix,  S.  365. 

3  Ann.  de  VInst.  Pasteur,  Paris,  1890,  t.  iv,  p.  570. 


Immunity  against  pathogenic  micro-organisms    145 

reaction  is  very  slight  or  absent.  To  corroborate  this  general  con- 
clusion, Wagner,  instead  of  lowering  the  temperature  by  means  of 
cold  water,  made  use  of  antipyrin  and  chloral.  The  application  of 
this  treatment  likewise  caused  enfeeblement  of  the  natural  defence 
of  the  organism  and  suppressed  the  immunity  of  the  fowl  against 
anthrax. 

Trapeznikoff1  has  studied  carefully  the  fate  of  anthrax  spores 
when  injected  into  fowls.  He  observed  that  most  of  them  are 
devoured  by  the  leucocytes.  Some  of  the  spores  were  first  trans-  [154] 
formed  into  small  rods,  sometimes  growing  into  real  bacilli,  but 
finally  they  all  became  the  prey  of  phagocytes  and  perished  in 
their  interior.  Those  in  the  vegetative  condition  are  soon  digested, 
the  spores,  however,  persist  for  some  time  inside  the  phagocytes, 
but  ultimately  disappear.  The  phagocytosis  in  fowls  inoculated 
with  spores  is  very  marked,  and  preparations,  stained  by  Ziehl's 
method,  demonstrate  most  clearly  the  reality  of  this  reaction  pheno- 
menon. These  preparations  have  for  long  been  used  in  the  course 
in  bacteriology  at  the  Pasteur  Institute  for  the  demonstration  of 
phagocytosis. 

In  the  face  of  these  facts,  well  established  and  confirmed  many 
times,  it  is  impossible  to  accept  Thiltges' 2  denial  of  the  ingestion  of 
these  bacteria  by  the  phagocytes  of  the  fowl.  Some  fault  of  technique, 
which  I  am  not  at  the  moment  in  a  position  to  indicate  exactly, 
has  evidently  slipped  into  this  author's  work.  The  positive  data, 
however,  on  phagocytosis  in  the  fowl,  obtained  by  Hess,  Wagner,  and 
Trapeznikoff,  data  confirmed  by  myself,  render  unnecessary  any  fresh 
researches  for  the  purpose  of  explaining  the  negative  results  obtained 
by  Thiltges.  As  regards  his  experiments  on  the  bactericidal  action 
of  defibrinated  blood  and  of  blood  serum  of  fowls  against  the  bacillus 
and  its  spores,  experiments  whose  results  are  opposed  by  those  of 
Wagner,  the  contradiction  may  be  explained  pretty  easily,  at  least  in 
part.  Thiltges  mentions  several  times  that  the  bacilli,  when  sown 
in  the  blood  serum  of  the  fowl,  were  aggregated  in  clumps.  Never- 
theless, he  has  failed  to  guard  against  this  source  of  error  and  has 
attributed  the  diminution  in  number  of  the  colonies  on  plates  to 
the  destruction  and  not  to  the  agglutination  of  the  bacilli.  Thiltges 
gives  so  few  particulars  of  the  conditions  under  which  his  experi- 
ments were  performed  that  we  do  not  even  know  at  what  temperature 

1  Ann.  de  Flnst.  Pasteur,  Paris,  1891,  t.  v,  p.  362. 

2  Ztschr.f.  Hyg.,  Leipzig,  1898,  Bd.  xxvm,  S.  189. 

B.  10 


146  Chapter  VI 

he  kept  his  tubes  containing  blood  and  serum  sown  with  bacilli.  As 
Wagner  kept  his  at  42° — 43°  C.,  a  temperature  which  corresponds  to 
that  of  the  body  of  the  fowl,  I  asked  M.  Gengou  to  make  a  series  of 
experiments  on  the  bactericidal  power  of  the  plasma  and  blood 
serum  of  fowls  on  the  anthrax  bacillus,  keeping  his  tubes  at  37°  C. 
[155]  The  result  of  his  experiments  was  in  complete  accord  with  those  of 
Wagner.  Under  the  conditions  that  I  have  just  stated  the  fluids 
of  the  fowl  are  no  more  bactericidal  than  they  are  under  the  con- 
ditions maintained  in  Wagner's  experiments. 

In  summing  up  these  data  on  the  natural  immunity  of  fowls 
against  anthrax,  we  are  certainly  justified  in  concluding  that  it  is 
due  to  the  phagocytosis  and  not  to  any  bactericidal  property  of  the 
"humours." 

The  pigeon  is  more  susceptible  than  the  fowl  to  the  action  of 
the  anthrax  bacillus,  still  it  manifests  a  certain  degree  of  resistance 
against  the  microbe.  After  what  we  have  said  on  the  subject  of 
the  fowl  we  need  make  but  few  remarks  on  the  pigeon,  in  spite  of 
the  very  animated  discussions  that  have  taken  place  on  the  mechan- 
ism of  its  immunity.  When  Baumgarten  was  offering  a  systematic 
opposition  to  the  part  played  by  phagocytic  reaction  in  immunity, 
he  set  his  pupil  Czaplewski1  to  investigate  the  resistance  of  pigeons 
against  anthrax.  The  results  of  this  investigation  were  absolutely 
negative  as  regards  phagocytosis.  The  latter  was  said  to  have  no 
importance  in  the  defence  of  the  organism,  which  resisted  simply 
because  it  was  impossible  for  the  bacillus  to  live  in  the  body  of 
the  pigeon.  I  then  set  myself  to  study  this  question2,  and  I  was 
able  to  demonstrate  that  the  anthrax  bacillus  is  quite  capable  of 
keeping  alive  in  the  pigeon,  that  it  can  develop  in  its  fluids,  but 
that  it  is  unable  to  defend  itself  against  the  aggression  of  the  phago- 
cytes which  ingest  it  and  completely  digest  it.  By  isolating  the 
phagocytes  that  had  ingested  the  bacilli  injected  into  the  body  of  the 
pigeon,  I  was  able  to  prove  that  a  number  of  these  bacilli  were  still 
alive.  The  enfeeblement  and  death  of  the  phagocytes  when  outside 
the  body  allowed  the  anthrax  bacilli  again  to  get  the  upper  hand 
in  this  struggle,  to  develop  and  to  give  virulent  cultures.  The  part 
played  by  phagocytes  in  this  example  of  natural  immunity  was  thus 
placed  beyond  doubt. 

1  "  Untersuchungen  ii   die  Immunitat  d.  Tauben,"  Konigsberg,  1889;  Ziegler's 
Beitr.  z.  path.  Anat.,  Jena,  1890,  Bd.  vn,  S.  49. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1890,  t.  iv,  p.  38 ;  p.  65. 


Immunity  against  pathogenic  micro-organisms    147 

Later,  Czaplewski1  himself  became  convinced  that  his  previous 
negative  results  would  not  stand  criticism,  and  Thiltges,  in  his  work 
already  mentioned,  when  discussing  the  fowl,  was  able  to  confirm  the  [156] 
importance  of  phagocytosis  in  the  defence  of  the  organism  of  the 
pigeon  against  anthrax.  He  was  struck  by  the  difference  between 
these  two  species  of  birds.  In  the  pigeon  it  was  easy  for  him  to 
prove  that  in  the  individuals  that  succumb  to  anthrax  the  phagocytic 
reaction  is  very  feeble,  whilst  in  those  which  ultimately  resist  the 
bacillus  it  is  very  pronounced.  Thiltges  likewise  observed  that  the 
blood  and  blood  serum  of  pigeons  when  sown  in  vitro  with  the 
anthrax  bacillus,  manifest  only  an  insignificant  bactericidal  power, 
a  fact  that  further  warrants  him  in  attributing  great  importance  to 
phagocytosis  in  the  maintenance  of  the  natural  immunity  of  the 
pigeon.  It  is  remarkable  that,  in  presence  of  these  facts,  it  did  not 
occur  to  the  author  to  ask  whether  this  fundamental  difference  in  the 
mechanism  of  the  resistance,  which  he  thought  possible  in  two  birds 
so  closely  allied  as  are  the  pigeon  and  the  fowl,  really  did  exist  in 
nature.  I  infer  that  his  experiments  on  the  fowl  were  made  before 
those  on  the  pigeon,  and  that  the  difference  in  his  results  depended 
specially  on  the  fact  that  he  had  acquired  greater  skill  in  executing 
his  later  experiments. 

Having  observed  that  frogs  die  readily  when  inoculated  with  an 
anthrax  bacillus  that  was  adapted  to  develop  at  a  low  temperature, 
Dieudonne  (I.e.)  endeavoured  to  suppress  the  immunity  of  the  pigeon 
by  using  bacilli  adapted  to  a  high  temperature.  But  the  inoculation 
of  a  second  generation  of  the  anthrax  bacillus,  cultivated  at  42°  C., 
was  borne  by  five  pigeons  without  inconvenience.  Even  bacilli 
that  were  rendered  capable,  by  cultivation  through  sixteen  genera- 
tions, of  developing  at  this  temperature  were  not  in  a  condition 
to  kill  more  than  five  pigeons  out  of  thirteen  inoculated.  These 
attempts  to  explain  immunity  as  due  to  the  properties  of  the  bacilli 
rather  than  to  those  of  the  organism  of  the  pigeon,  have  therefore 
led  to  a  result  very  different  from  that  anticipated  by  Dieudonne. 

The  pigeon  is  further  of  interest  to  us  because  of  its  natural 
immunity  against  the  bacillus  of  human  tuberculosis.  It  resists 
considerable  doses  of  this  bacillus,  so  virulent  for  man  and  for  the 
majority  of  mammals,  and  even  for  some  birds  (canaries  and  parrots). 
Dembinski2,  studying  the  mechanism  of  this  immunity,  was  able  to 

1  Ztschr.f.  Hyg.,  Leipzig,  1892,  Bd.  xn,  S.  348. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1899,  t.  xiu,  p.  426. 

10—2 


148 


Chapter  VI 


prove  that  the  bacilli  of  human  tuberculosis  encounter  in  the  organ- 
ism of  the  pigeon  a  very  great  resistance  from  the  phagocytes, 
[157]  especially  from  the  macrophages.  These  cells  fuse  together  around 
masses  of  bacilli  and  imprison  them  within  real  giant  cells  or  poly- 
nucleated  macrophages  (Fig.  21).  The  inicrophages  in  this  struggle 
play  only  a  secondary  part, 
but  the  resistance  offered  by 
the  macrophages  is  a  most 
effective  one.  Incapable  of 
completely  destroying  the 
bacilli,  these  phagocytes 
exercise  over  them  an  un- 
favourable influence  and 
prevent  them  from  multi- 
plying and  exhibiting  their 
noxious  action.  The  im- 
portance of  the  defence  by 
the  macrophages  comes  out 
still  more  clearly  when  com- 
pared with  what  takes  place 
if,  instead  of  the  bacillus 
of  human  tuberculosis,  we 
inoculate  into  pigeons  the 
bacillus  of  avian  tubercu- 
losis. In  the  latter  case 
the  microphages  certainly 
promptly  seize  the  bacilli, 

but  being  powerless  against  them  they  perish,  whilst  the  macrophages 
only  intervene  later  on  and  in  small  numbers.  The  result  is  that 
in  the  pigeon  the  avian  bacillus  becomes  generalised  in  the  organism 
and  sets  up  a  fatal  tuberculosis. 

It  must  be  admitted,  then,  that  the  immunity  of  the  pigeon  against 
the  bacillus  of  human  tuberculosis  is  due  to  the  defence  by  the 
macrophages.  This  conclusion  is  corroborated  by  the  fact  that  in 
the  fowl — equally  refractory  against  the  same  bacillus — there  is  also 
observed  a  very  strong  macrophagic  reaction. 

Nocard1,  who  for  several  years  has  been  carrying  on  studies  on 
the  relations  between  the  bacilli  of  human  and  avian  tuberculosis, 
conceived  the  idea  of  adapting  the  former  to  the  organism  of  the 
1  Ann.  de  VInst.  Pasteur,  Paris,  1898,  t.  xil,  p.  561. 


T-    f 


FIG.  21.  Reaction  of  the  phagocytes  of  the 
pigeon  against  the  bacilli  of  human  tuber- 
culosis. 


Immunity  against  pathogenic  micro-organisms    149 

fowl.  With  this  object  he  enclosed  a  culture  of  the  bacillus  of  [158] 
human  tuberculosis  in  a  sac  of  collodion  which  he  then  introduced 
into  the  peritoneal  cavity  of  fowls.  Under  these  conditions  the 
bacillus,  protected  against  the  aggression  of  phagocytes,  continued 
to  live  inside  the  sac  through  whose  walls  the  fluid  part  of  the 
peritoneal  lymph  could  diffuse.  After  several  passages  from  sac  to 
sac  the  human  bacillus  becomes  acclimatised  to  the  body  of  the  fowl 
and  is  transformed  into  a  variety  quite  comparable  to  the  bacillus 
of  avian  tuberculosis.  This  experiment  has  definitely  settled  the 
question  so  long  under  discussion  of  the  specific  difference  between 
the  two  tubercle  bacilli.  It  has  resolved  it  in  the  sense  of  affirming 
their  unity;  the  avian  bacillus  is  only  a  modified  race  of  the  same 
bacillus  which  sets  up  tuberculosis  in  man  and  other  mammals. 

In  spite  of  the  great  difference  between  the  anthrax  bacillus  and 
that  of  human  tuberculosis,  the  immunity  against  these  two  bacteria, 
which  is  shown  in  birds,  depends  in  every  case  upon  the  reaction  of 
the  phagocytic  system. 

Having  rapidly  glanced  at  natural  immunity  as  we  ascend  the 
scale  of  the  animal  series  we  now  come  to  it  as  it  presents  itself  in 
the  highest  class,  Mammals,  a  question  on  which  it  is  necessary  to 
dwell  at  greater  length  because  of  its  great  importance,  and  also 
because  of  the  fuller  study  that  has  been  given  to  it. 

As  the  immunity  of  the  Invertebrata  and  of  the  lower  Vertebrata 
against  the  anthrax  bacillus  has  furnished  us  with  several  important 
indications  we  will  first  endeavour  to  throw  light  on  the  mechanism 
of  the  resistance  offered  to  anthrax  by  certain  mammals.  The  repre- 
sentatives of  this  class  being,  however,  for  the  most  part  extremely 
susceptible  to  this  disease,  examples  of  true  natural  immunity  are 
very  rare.  The  first  place  among  resistant  mammals  is  occupied  by 
the  dog.  Although  young  dogs,  as  demonstrated  by  Strauss1,  readily 
take  fatal  anthrax,  the  canine  species  may  nevertheless  be  regarded 
as  possessing  a  real  immunity,  as  adult  dogs  withstand,  without 
inconvenience,  the  inoculation  of  large  quantities  of  bacilli.  When 
introduced  beneath  the  skin  these  bacilli  excite  a  local  inflammation, 
accompanied  by  a  very  marked  diapedesis  of  white  corpuscles  which  at 
once  begin  to  devour  the  bacilli.  This  phagocytosis  has  already  been 
observed  by  Hess2,  Malm3,  myself,  and  several  other  investigators,  [159] 

1  Arch,  de  med.  exper.  et  danat.  path.,  Paris,  1889, 1. 1,  p.  325. 

2  VirchrjvJs  Archiv,  Berlin,  18S7,  Bd.  cix,  S.  365. 

3  Ann.  de  llnst.  Pasteur,  Paris,  1890,  t.  iv,  p.  520. 


150  Chapter  VI 

so  that  its  existence  cannot  be  doubted.  Recently,  Martel1  has 
demonstrated  a  very  distinct  phagocytic  reaction  in  all  those  cases 
where  he  has  had  to  deal  with  dogs  that  were  refractory  or  not  very 
susceptible.  This  reaction  is  shown  by  the  ingestion  of  the  bacteria 
and  by  the  large  accumulation  of  leucocytes  at  the  seat  of  inocu- 
lation. His  researches  are  of  special  interest  by  reason  of  the 
counter-test  that  he  was  able  to  make  upon  dogs  that  were 
susceptible  to  anthrax.  It  was  demonstrated  some  years  ago  that 
the  natural  immunity  of  the  dog  against  the  bacillus,  although  very 
real,  is,  nevertheless,  relative  and  limited.  Thus  Bardach2  established 
the  fact  that  dogs  from  whom  the  spleen,  an  organ  full  of  phagocytes, 
had  been  removed,  became  susceptible  to  anthrax.  Even  dogs  into 
whose  veins  he  injected  fine  wood-charcoal  powder  suspended  in 
water,  with  the  purpose  of  "diverting"  the  phagocytosis,  readily 
succumbed  to  anthrax. 

Martel  endeavoured  to  suspend  the  natural  immunity  of  dogs  by 
injecting  into  them  phloridzin  or  pyrogallic  acid.  But  he  obtained 
much  more  constant  results  by  inoculating  the  bacillus  into  rabid 
dogs.  The  organism,  weakened  by  this  terrible  disease,  became 
very  susceptible  to  anthrax,  and  the  rabid  animal  succumbed  to 
anthrax  before  the  rabies  had  completed  its  evolution.  By  its 
passage  through  the  rabid  dog  the  anthrax  virus  is  so  augmented 
in  virulence  that  it  becomes  fatal  for  normal  dogs.  Martel  succeeded 
also  in  reinforcing  the  bacillus  isolated  from  a  cow  affected  with 
anthrax.  In  all  these  cases  where  the  reinforced  bacilli  set  up  a 
severe  and  rapidly  fatal  infection,  Martel  could  demonstrate  only 
a  feeble  phagocytic  reaction. 

Researches  on  the  phagocytosis  of  dogs,  inoculated  with  the 
anthrax  bacillus,  have  always  demonstrated  a  regular  and  constant 
relation  between  this  reaction  and  the  resistance  of  the  organism. 
On  the  other  hand,  experiments  undertaken  for  the  purpose  of 
establishing  the  part  played  by  the  body  fluids  in  this  immunity, 
have  always  given  negative  results. 

As  the  dog,  of  all  mammals,  exhibits  the  greatest  natural  im- 
munity from   anthrax,   it  is  very  natural  that  in  the  bactericidal 
property  of  its  blood  the  key  to  the  enigma  has  been  sought.    Thus 
[loo]  Nuttall3  concludes  from  his  experiments  that  the  anthrax  bacillus 

1  Ann.  de  VInst.  Pasteur,  Paris,  1900,  t.  xiv,  p.  13. 

2  Ann.  de  FInst.  Pasteur,  Paris,  1889,  t.  in,  p.  577. 

3  Ztschr.f.  Hyg.,  Leipzig,  1888,  Bd.  iv,  S.  353. 


Immunity  against  pathogenic  micro-organisms    151 

is  readily  destroyed  by  defibrinated  dog's  blood.  But,  as  this  result 
was  not  in  accord  with  my  observations1  that  the  bacillus  is  easily 
cultivated  in  dog's  blood,  and  as  several  observers,  especially 
Lubarsch2,  had  arrived  at  conclusions  opposed  to  those  of  Nuttall, 
systematic  researches  were  made  for  the  purpose  of  solving  this 
complicated  problem.  Denys  and  Kaisin3  sought  to  remove  the 
objections  formulated  against  the  explanation  of  the  immunity  of 
the  dog  as  due  to  the  bactericidal  property  of  its  blood  by  affirm- 
ing that  this  power,  which  is  absent  in  the  inoculated  dog,  develops 
whilst  the  animal  is  under  the  influence  of  the  bacillus.  Immunity 
is  reduced,  then,  in  this  case  to  the  establishment  of  a  new 
property  in  the  fluids  during  the  course  of  the  struggle  of  the 
organism  against  the  inoculated  bacillus.  Xone  of  the  observers, 
however,  who  have  repeated  these  experiments,  e.g.  Lubarsch4  and 
Bail5,  were  able  to  confirm  the  results  of  the  Belgian  observers. 
Denys  himself,  indeed,  having  resumed  this  study  with  Havet6, 
had  to  reject  the  conclusions  of  his  former  work  executed  in 
collaboration  with  Kaisin.  He  is  persuaded  that  their  error  was 
due  to  the  fact  that  in  their  experiments  in  vitro,  the  living  leuco- 
cytes ingested  the  bacilli  and  prevented  their  development.  As  the 
result  of  these  new  researches  Deuys  and  Havet  have  come  to  the 
conclusion  "  that  the  main,  the  predominating  part  of  the  bactericidal 
power  of  the  dog's  blood  must  be  ascribed  to  the  leucocytes  acting  as 
phagocytic  elements"  (loc.  cit.  p.  15). 

As  a  result  of  the  investigations  I  have  summarised  the  conclusion 
is  forced  upon  us  that  the  natural  immunity  of  the  dog  from  anthrax 
is  a  function  of  the  phagocytes.  In  presence  of  this  uniformity  of  the 
experimental  results  it  becomes  very  important  to  make  a  more  pro- 
found study  of  the  phenomena  that  manifest  themselves  during  the 
destruction  of  the  bacilli  by  the  phagocytes  of  the  dog.  What  are  the 
phagocytic  elements  which  play  the  principal  part  in  this  struggle, 
and  by  what  means  do  they  attain  this  result  ?  Gengou7  undertook  a  [161] 

1  Ann.  de  Vlnst.  Pasteur,  Paris,  1887,  t.  I,  p.  43. 

2  "  Untersuchimgen  ii.  die  Ursachen  der  angeborenen  u.  erworbenen  Immunitat," 
Berlin,  1891,  S.  111. 

3  La  Cellule,  Lierre  et  Lou  vain,  1893,  t.  ix,  p.  337. 

4  "Zur  Lehre  von  den  Geschswiilsten  und  Infectionskrankheiten,"  Wiesbaden,  1899. 

5  CentralU.  f.  Bacterial,  u.  Parasitenk.,  lte  Abt.,  Jena,  1900,  Bd.  xxvn,  SS.  10 
und  517. 

6  La  Cellule,  Lierre  et  Louvain,  1894,  t.  x,  p.  7. 

7  Ann.de  Vlnst.  Pasteur,  Paris,  1901,  t.  xv,  p.  68. 


152  Chapter  VI 

detailed  investigation  in  my  laboratory  to  answer  these  questions. 
He  was  able  to  convince  himself,  in  agreement  with  the  statements 
of  his  predecessors,  that  not  only  was  the  serum  of  dog's  blood  not 
bactericidal  for  the  anthrax  bacillus,  but  that  the  plasma  of  the  blood 
is  no  more  so.  The  fluid  of  the  aseptic  pleural  exudation  obtained 
after  injection  of  gluten-casein,  was  likewise  incapable  of  killing  the 
anthrax  bacillus.  When  Gengou,  by  means  of  centrifugalisation, 
isolated  the  leucocytes  from  these  exudations,  washed  them  in 
physiological  salt  solution,  froze  them,  and  then  macerated  them  in 
broth,  he  obtained  suspensions  of  white  corpuscles,  to  which  he 
added  bacilli.  He  was  able  to  demonstrate  that  when  the  exudations 
contained  macrophages  principally,  as  is  observed  in  exudations 
taken  at  the  end  of  two  or  three  days,  the  bactericidal  power  of 
the  suspensions  was  nil  or  insignificant.  When,  on  the  other  hand, 
the  leucocytes  came  from  exudations  only  twenty- four  hours  old  and 
were  composed  almost  exclusively  of  microphages,  the  destructive 
action  on  the  bacilli  of  the  extract  of  the  microphages  in  broth 
was  most  marked.  Now  it  is  fully  demonstrated  that  in  the 
exudation  set  up  in  the  refractory  dog  by  the  injection  of  anthrax 
bacilli,  it  is  especially  the  microphages  which  exhibit  the  phagocytic 
reaction  against  this  bacillus. 

This  is  how  the  question  of  the  immunity  of  the  dog  from 
anthrax  stands  at  present.  The  natural  immunity  of  this  species, 
which  although  not  unlimited,  is  very  real,  depends  on  the  activity 
of  phagocytes.  These  elements,  under  the  stimulus  of  the  bacillus 
and  its  products,  exhibit  a  positive  chemiotaxis  of  the  most  marked 
character,  they  approach  the  bacilli,  ingest  them  by  a  physiological 
act,  and  destroy  them  by  means  of  a  substance  which  is  not  found  in 
either  the  plasma  or  the  blood  serum,  but  which  can  be  demonstrated 
in  an  extract  of  the  microphages. 

In  spite  of  the  uniformity  and  precision  of  these  data,  it  is 
impossible  to  rest  satisfied  with  describing,  as  an  example  of  natural 
immunity  from  anthrax,  the  single  case  of  the  dog.  If  the  resistance 
of  the  rat  against  this  disease  was  merely  of  historical  interest  because 
of  the  large  number  of  works  devoted  to  this  question,  we  might 
[162]  relegate  it  to  the  chapter  reserved  for  the  history  of  our  knowledge 
on  immunity.  But  it  is  not  so.  The  anthrax  of  rats  is  a  subject 
full  of  very  valuable  instruction,  and  von  Behring  was  quite  justified 
in  saying  that  whoever  wished  to  get  a  true  conception  of  natural 
immunity  from  a  virus  should  pay  special  attention  to  this  example. 


Immunity  against  pathogenic  micro-organisms    153 

As  a  matter  of  fact,  it  may  be  stated  that  the  grey  rat  (Mus 
decumanus),  the  black  rat  (Mus  rattus\  and  white  rats  are  far  from 
enjoying  a  true  immunity  from  anthrax.  They,  nevertheless,  exhibit 
a  more  or  less  marked  resistance  against  this  disease  and  are  always 
less  susceptible  than  are  the  other  laboratory  rodents  :  mice,  guinea- 
pigs  and  rabbits.  Rats  resist  attenuated  bacilli  (anthrax  vaccines) 
better  than  do  these  three  species,  and  in  order  to  induce  in  them 
fatal  anthrax  it  is  necessary  to  inoculate  a  much  larger  number  of 
virulent  bacilli.  On  the  other  hand,  rats  are  distinguished  by  a  great 
irregularity  in  the  resistance  they  offer  to  the  bacillus.  At  times 
they  resist  very  virulent  bacilli ;  at  others  they  contract  a  fatal 
disease  after  an  injection  of  very  attenuated  bacilli  (Pasteur's  first 
vaccine). 

In  my  first  memoir  on  anthrax1  I  noted  the  fact  that  in  rats  the 
phagocytosis  against  the  bacillus  when  injected  subcutaneously  was 
more  marked  than  after  the  same  inoculation  into  the  rabbit  and 
guinea-pig.  Later,  this  fact  was  disputed  by  several  observers,  who 
refused  to  accept  the  extent  and  importance  of  the  phagocytic  reaction 
in  the  rat.  This  opposition  was  strengthened  by  a  very  interesting 
discovery  made  by  von  Behring2,  namely,  that  the  blood  serum  of 
the  rat  possessed  a  remarkably  destructive  power  for  the  anthrax 
bacillus.  When  this  observer  added  a  certain  quantity  of  anthrax 
bacilli  to  some  blood  serum  of  the  rat,  instead  of  elongating  into 
filaments  and  dividing  they  underwent  a  change,  lost  their  normal 
refraction  and  took  on  staining  reagents  very  imperfectly.  The 
membranes  alone  remained  of  the  bacilli  thus  treated.  Von  Behring 
considered  that  this  bactericidal  action  of  the  serum  depends  on 
the  presence  of  an  organic  base  dissolved  in  the  blood  fluid.  He 
had  merely  to  neutralise  the  serum  by  means  of  an  acid,  and  there 
was  at  once  a  very  abundant  development  of  the  bacillus.  From 
these  researches  von  Behring  came  to  the  conclusion  that  the 
natural  immunity  of  the  rat  from  anthrax  can  be  reduced  to  terms  [163] 
of  the  chemical  action  of  the  blood  on  the  bacillus. 

In  one  of  his  most  recent  publications  this  author3  returns  to  the 
question  of  anthrax  in  rats  and  sums  up  his  present  point  of  view 
as  follows.  He  regards  the  immunity  of  these  rodents  as  being 

1  Virchow's  Archiv.  Berlin,  1884,  Bd.  xcvn,  S.  516. 

2  Centralblf.  Jdin.  Med.,  Bonn,  1888,  Xo.  38. 

3  'l  Infectionsschutz  und  Immunitat "  in  Eulenberg's  "  Real-Encyclopadie  d.  ges. 
Heilkunde/'  mte  Aufl.  (Encyclop.  Jfrfafytofrffr),  Wien,  1900,  Bd.  ix,  S.  196. 


154  Chapter  VI 

relative,  not  absolute.  "The  anthrax  bacilli"— he  says— "die  in  rat's 
serum  in  vitro ;  and  in  the  cases  where  the  inoculation  of  these 
animals  with  the  anthrax  virus  is  not  fatal,  it  is  at  least  reasonable  to 
assume  that  the  blood  fluid  likewise  produces  this  protection  in 
the  organism  of  the  living  rat.  Now,  an  immunity  that  manifests 
itself  without  the  aid  of  any  activity  of  the  cell  must  undoubtedly 
be  regarded  as  being  of  a  humoral  character  "  (Joe.  cit.  p.  202). 

Let  us  begin  by  analysing  the  facts  as  presented  in  rats  into 
[164]  whose  subcutaneous  tissue  we  have  injected  anthrax  virus.  A  certain 
number  of  them  resist,  without  exhibiting  any  lesion  other  than 
a  certain  exudative  inflammation  at  the  seat  of  inoculation.  The 
exudation  is,  in  this  case,  very  rich  in  leucocytes  which  quickly 
exert  their  phagocytic  function  and  destroy  the  ingested  bacilli. 
In  this  reaction  it  is  the  microphages  that  play  the  chief  part,  the 
macrophages  intervening  later  and  in  a  much  less  pronounced  fashion. 
Usually,  however,  the  inoculated  rats  exhibit  a  more  serious  illness : 
the  bacilli  multiply  at  the  point  of  inoculation  and  excite  the 
formation  of  an  extensive  oedema,  rich  in  serous  fluid,  transparent, 
and  very  poor  in  leucocytes.  It  is  only  later  that  these  cells  inter- 
vene in  any  considerable  number.  The  exudation  becomes  thicker 
and  turbid,  the  numerous  white  corpuscles  devour  the  bacilli  and 
cause  their  disappearance.  Under  the  influence  of  this  marked 
reaction  the  animals  in  most  cases  recover,  as  has  already  been 
established  by  Frank1.  But  even  in  those  individuals  which  succumb 
to  anthrax  death  occurs  more  or  less  tardily,  an  examination  of  the 
internal  organs  then  revealing  a  considerable  phagocytic  reaction. 
The  spleen,  often  of  enormous  size,  contains  numerous  macrophages 
which  are  filled  with  normal  or  more  or  less  altered  bacilli.  In  the 
liver  these  macrophages,  which  have  devoured  several  microphages 
and  some  bacteria,  are  also  found  (Figs.  22  and  23). 

When  instead  of  bacteria  in  the  condition  of  rods,  anthrax  spores 
are  inoculated  subcutaneously  or  into  the  anterior  chamber  of  the 
eye,  we  can  observe  their  germination.  There  is  developed  a  whole 
generation  of  bacilli  which  behave  like  those  we  have  already 
described,  that  is  to  say,  they  excite  an  exudation  and  are  ultimately 
digested  within  the  phagocytes  (Figs.  24  and  25).  All  these  pheno- 
mena of  phagocytosis  I  described  in  detail  more  than  ten  years  ago 
in  my  memoir  on  the  anthrax  of  rats2.  Since  then  not  a  single 

1  Centralblf.  Bacteriol  u.  Parasitenk.,  Jena,  1888,  Bd.  iv,  SS.  710,  737. 

2  Ann.  de  I'Inst.  Pasteur,  Paris,  1890,  t.  IV,  p.  193. 


Immunity  against  pathogenic  micro-organisms    155 

fact  has  been  brought  forward  to  invalidate  the  results  there  set 

forth. 


FIG.  23.  Macrophage  containing 
bacilli,  from  the  liver  of  a  rat 
affected  with  anthrax. 


PIG.  22.     Macrophage  from  the  liver  of 
a  rat  affected  with  anthrax. 


How  is  this  paradoxical  fact  to  be  explained,  that  anthrax  which 
grows  in  the  body  of  the  rat,  there  setting  up  a  disease  more  or  less 
grave  and  sometimes  fatal,  is  so  readily  destroyed  by  the  serum  and 


FIG.  24.     Microphage  of  rat 
filled  with  bacilli. 


FIG.  25.  Two  micro  - 
phages  of  rat  that 
have  ingested  bacilli. 


156  Chapter  VI 

blood  when  removed  from  the  organism?  From  numerous  experi- 
[165]  ments,  carried  out  by  Hankin1  and  by  Roux  and  myself2,  it  has  been 
demonstrated  that  the  bactericidal  power  of  the  fluids  of  the  rat 
cannot  be  invoked  as  the  cause  of  the  animal's  resistance  to  anthrax. 
Those  rats  which  show  themselves  very  susceptible  to  this  disease  and 
die  from  anthrax  infection,  furnish,  nevertheless,  a  serum  that  will 
prevent  anthrax  in  other  rats,  and  which  will  protect  even  mice  into 
which  the  bacilli  have  been  injected.  Rats  into  which  we  inoculate 
on  one  side  of  the  body  a  little  anthrax  culture,  and  on  the  other 
side  the  same  quantity  of  bacilli  mixed  with  blood  serum  from  the 
same  animal,  manifest  oedema  at  the  former  place  only.  It  is  from 
this  latter  point  that  the  general  infection  takes  place,  the  side  where 
the  anthrax  bacilli  mixed  with  serum  was  introduced  remaining 
unaffected.  Sawtchenko3,  who  has  investigated  the  immunity  of  the 
rat  in  my  laboratory,  has  to  the  facts  just  mentioned  added  the 
observation  that  when  the  injection  of  bacilli  causes  haemorrhage 
the  rat  survives.  When,  on  the  contrary,  the  injection  is  made  with 
a  fine  needle  and  without  effusion  of  blood,  the  rat  contracts  a 
fatal  anthrax. 

It  follows  from  these  facts  that  the  blood,  immediately  it  has 
[166]  escaped  from  the  vessels,  undergoes  a  change  in  its  composition  and 
becomes  bactericidal  for  the  anthrax  bacillus,  whilst,  when  it  is  circu- 
lating in  the  organism,  it  exhibits  no  such  power.  Sawtchenko  has 
studied  the  substance  in  the  serum  which  kills  the  bacilli  and  has 
demonstrated  that  it  will  resist  heating  to  56°  C.;  even  when  heated 
to  61  °C.  the  serum  still  exercises  a  certain  amount  of  bactericidal 
power  for  very  attenuated  bacilli  (Pasteur's  first  vaccine).  Researches 
on  the  distribution  of  this  bactericidal  power  in  the  living  rat  have 
convinced  Sawtchenko  that  none  of  it  passes  into  the  fluid  of  the 
passive  oedema  set  up  by  the  slowing  of  the  circulation,  nor  into  that 
of  the  active  oedema  developed  as  the  result  of  the  inoculation  of 
anthrax  bacilli.  He  observed  that  even  the  bacillus  of  Pasteur's  first 
vaccine  grows  abundantly  in  the  oedematous  fluid  produced  by  the 
injection  of  virulent  bacilli.  The  peritoneal  lymph,  however,  exerts 
a  very  marked  bactericidal  action  on  the  bacilli.,  Having  demon- 
strated this  fact  Sawtchenko  put  to  himself  the  question :  May  not 
the  great  difference  between  the  action  of  these  fluids  depend  on  the 

1  CentralU.f.  Bacterial,  u.  Parasitenk.,  Jena,  1891,  Bd.  ix,  SS.  336,  372. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1891,  t.  v,  p.  479. 

3  Ann.  de  VInst.  Pasteur,  Paris,  1897,  t.  xi,  p.  865. 


Immunity  against  pathogenic  micro-organisms    157 

fact  that  the  lymph  is  rich  in  leucocytes,  whilst  in  the  fluid  of  the 
oedema  they  are  almost  absent  ?  Pursuing  this  question,  Sawtchenko 
made  a  comparative  study  of  the  bactericidal  power  of  the  serum, 
prepared  outside  the  body,  and  of  the  blood  plasma  obtained  by 
means  of  an  extract  of  the  heads  of  leeches,  and  he  concluded 
from  his  researches  that  the  bactericidal  substance  circulates  in  the 
plasma  of  the  living  rat  and  that  it  is  not  derived  from  the  micro- 
phages,  but  must  be  looked  upon  rather  as  a  secretion  of  the  macro- 
phages  in  the  blood  and  of  endothelial  cells.  This  result  was  not 
confirmed  by  Gengou1,  who  also  took  up  the  study  of  this  important 
question  in  my  laboratory.  Instead  of  preparing  the  plasma  by 
means  of  the  addition  of  an  extract  of  leeches  he  made  use  of 
a  method  much  more  perfect  and  free  from  sources  of  error.  He 
introduced  no  foreign  substance  capable  of  affecting  the  results  of  his 
experiments.  Collecting  the  rat's  blood  in  paraffined  tubes,  and 
centrifugalising  it  in  similar  tubes,  he  obtained  a  fluid  which  ap- 
proaches much  more  closely  the  plasma  of  circulating  blood  than  does 
serum.  This  fluid,  however,  will  coagulate  at  the  end  of  a  fairly  long 
interval,  which  proves  that  it  cannot  be  looked  upon  as  blood  plasma. 
Gengou  examined  the  bactericidal  power  of  the  fluid  portion  of  the 
"plasma,"  obtained  by  the  process  just  described,  on  the  anthrax [i 67] 
bacillus,  and  also  that  of  serum  prepared  in  tubes  in  the  ordinary 
way.  The  difference  between  the  two  fluids  is  very  marked ;  whilst 
the  serum  destroys  the  bacilli  sown  in  it  very  rapidly  and  dissolves 
their  contents,  the  fluid  of  the  "plasma"  has  no  similar  action. 
These  results,  confirmed  several  times,  demonstrate  very  definitely 
that  the  plasma  of  the  circulating  blood  does  not  contain  any  bac- 
tericidal substance.  This,  during  the  life  of  the  animal,  is  found 
inside  leucocytes  and  only  escapes  from  them  when  the  cells  burst 
or  undergo  profound  lesions,  this  taking  place  when  the  clot  is 
formed  and  when  the  serum  is  prepared  outside  the  organism,  or  in 
the  effused  and  coagulated  blood,  or  again  in  the  peritoneal  lymph 
during  phagolysis.  This  phagolysis  is  inevitably  produced  as  a  result 
of  rapid  injection  of  foreign  fluids  into  the  peritoneal  cavity,  e.g. 
of  broth  or  of  physiological  salt  solution,  containing  bacteria  in 
suspension. 

The  facts  we  have  brought  together  on  the  subject  of  anthrax  in 
rats  form  a  whole  whose  several  parts  are  in  complete  harmony.    The 
phagocytes  of  this  species  of  rodent  contain  a  bactericidal  ferment, 
1  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  232. 


158  Chapter  VI 

a  kind  of  cytase,  which  resists  temperatures  approaching  60°  C.  This 
cytase  is  very  active  against  the  bacilli,  but  in  the  living  animal  it 
can  only  act  within  the  phagocytes,  or,  in  a  transitory  and  incomplete 
fashion,  outside  these  cells,  when  phagolysis  is  taking  place  in  the 
peritoneal  cavity.  The  resistance  offered  by  the  rat  to  anthrax 
depends,  then,  on  this  phagocytic  activity.  For  its  manifestation  it  is 
necessary,  first  of  all,  that  the  phagocytes  should  manifest  a  positive 
chemiotaxis  for  the  bacilli,  and  then  that  they  should  seize  and  ingest 
these  organisms.  These  are  the  vital  acts  that  decide  the  result  of 
the  struggle.  When  the  phagocytes  show  themselves  inactive  the 
bacilli  multiply  in  the  oedematous  fluid  which  contains  no  bacteri- 
cidal cytase,  and  pass  into  the  plasmas  of  the  lymph  and  of  the  blood, 
which  also  are  incapable  of  killing  these  bacteria.  The  animal  may, 
then,  die  of  anthrax,  in  spite  of  the  presence  in  its  body  of  a  large 
quantity  of  bactericidal  cytase  which  is  to  be  found  in  situations  to 
which  the  bacilli  have  not  penetrated.  In  those  cases,  on  the  other 
hand,  where  the  phagocytes  accomplish  their  function,  where  they 
rush  up  to  the  menaced  point  and  devour  the  inoculated  bacteria, 
these  bacilli  are  placed  in  contact  with  the  intracellular  cytase  and 
[168]  undergo  complete  digestion.  The  organism  in  this  case  gets  rid  of  its 
enemies  and  victoriously  resists  infection. 

Anthrax  in  rats,  then,  presents  one  of  the  most  instructive 
examples  of  natural  immunity.  But  the  detailed  analysis  of  the 
mechanism  of  this  resistance  demonstrates  very  clearly  the  great 
part  played  by  the  phagocytes  in  this  process.  In  this  respect  the 
organism  of  the  rat  presents,  in  a  general  fashion,  a  great  analogy 
to  the  natural  immunity  of  the  dog,  of  birds,  and  of  other  repre- 
sentatives of  the  animal  kingdom  that  we  have  examined.  Under 
these  conditions  it  is  useless  to  insist  at  any  length  on  other  examples 
of  resistance  against  anthrax  which,  moreover,  have  relation  much 
more  often  to  a  natural  immunity  against  attenuated  bacilli  than  to 
one  against  true  anthrax  virus.  Rabbits  and  guinea-pigs,  so  sensitive 
to  this  virus,  often  resist  the  inoculation  of  Pasteur's  vaccines.  The 
rabbit  is,  in  general,  refractory  to  the  first  anthrax  vaccine ;  it  may 
even  resist  the  second  vaccine.  The  guinea-pig,  a  more  sensitive 
animal,  does  not  exhibit  any  natural  immunity  except  against  the 
first  vaccine.  In  all  these  cases  the  mechanism  is  similar  to  that 
which  the  rat  and  the  dog  oppose  to  virulent  anthrax.  The  bacilli, 
into  whatever  part  of  the  body  they  are  injected,  set  up  an  exudative 
inflammation  which  brings  up  a  large  number  of  leucocytes  to  the 


Immunity  against  pathogenic  micro-organisms     159 

point  menaced.  These  cells  readily  exert  their  phagocytic  function 
and  rid  the  organism  of  the  introduced  bacteria.  In  order  to  obtain 
a  complete  grasp  of  the  part  played  by  this  reaction  it  will  be  found 
useful  to  inject  beneath  the  skin  of  one  ear  of  a  rabbit  a  little  anthrax 
vaccine  and  beneath  the  skin  of  the  other  the  same  quantity  of  virulent 
bacilli.  The  difference  between  the  reaction  in  the  two  cases  is  very 
striking.  The  ear  inoculated  with  vaccine  soon  becomes  the  seat  of  a 
circumscribed  inflammation  with  a  purulent  exudation,  all  the  bacilli 
in  which  have  been  devoured  by  the  leucocytes.  The  other  ear,  on 
the  contrary,  presents,  around  the  injected  virus,  only  a  serous  or 
blood- tinged  exudation  containing  no,  or  few,  leucocytes  ;  the  bacilli 
are  found  free  in  the  liquid  and  multiply  without  let  or  hindrance. 
Meeting  with  no  opposition  the  virus  becomes  generalised  through- 
out the  organism  and  brings  on  death  by  anthrax  septicaemia. 
Rabbits,  into  which  anthrax  vaccines  only  are  introduced,  oppose  to 
the  invasion  of  the  bacilli  a  leucocytic  barrier  which  arrests  their 
extension.  The  natural  immunity  of  the  sheep,  rabbit  and  guinea- 
pig  is  also  a  phagocytic  immunity,  but  it  is  only  capable  of  being 
exercised  against  bacilli  previously  attenuated  in  virulence.  The  [169] 
researches  of  Mine  Metchnikoff1  on  the  reaction  of  the  phagocytes 
of  these  animals  to  the  bacilli  of  Pasteur's  two  anthrax  vaccines  have 
demonstrated  the  importance  of  the  destruction  of  these  bacilli  by 
the  leucocytes.  All  the  other  examples  of  natural  immunity  against 
anthrax  are  also  merely  relative.  The  fowl  that  resists  an  anthrax 
virus  strong  enough  to  kill  an  ox  or  a  horse,  succumbs  to  a  special 
variety  of  anthrax  cultivated  by  Levin2.  The  dog,  as  we  have  seen, 
in  spite  of  its  pronounced  natural  immunity  against  anthrax,  is  killed 
by  the  special  anthrax  bacillus  prepared  by  Martel. 

In  this  immunity  against  anthrax  we  have  to  deal  with  a  bacillus 
capable  of  living  and  reproducing  itself  in  extremely  varied  media. 
Hence  the  reason,  it  may  be  said,  that  the  bactericidal  influence 
of  the  fluids  is  so  little  pronounced  in  this  case.  To  bring  it  into 
relief  we  must,  therefore,  choose  a  bacterium  less  capable  of  adapting 
itself  to  the  chemical  composition  of  various  culture  media.  In  this 
matter  we  cannot  do  better  than  select  pathogenic  spirilla  of  ex- 
tremely delicate  nature  and  analyse  the  mechanism  of  the  natural 
immunity  of  certain  species  of  animals  with  respect  to  them.  It 
must  not  be  forgotten,  however,  that  here  we  are  making  use  of 

1  Ann.  de  VInst.  Pasteur,  Paris,  1891,  t.  v,  p.  145. 
9  l<  Om  Mjaltbrand  hos  Hons,"  Stockholm,  1897. 


160  Chapter  VI 

representatives  of  an  infinitely  small  minority  of  pathogenic  bacteria, 
the  majority  resembling  the  anthrax  bacillus  in  the  facility  with 
which  they  can  be  cultivated  in  all  sorts  of  nutritive  media. 

The  spirillum  of  recurrent  fever  of  man  (Spirochaete  obermeyeri} 
was  the  first  pathogenic  microbe  found  in  an  infective  disease 
distinctly  human.  Discovered  a  third  of  a  century  ago,  it  has  passed 
through  the  hands  of  the  most  skilful  bacteriologists,  who  have  tried 
all  possible  methods  of  cultivating  it  outside  the  body.  Koch  him- 
self tried  to  solve  the  problem,  but,  in  spite  of  his  incomparable 
skill,  did  not  succeed.  Later,  Sakharoff1,  at  Tiflis,  discovered  a 
spirillum  very  similar  in  appearance  which  produced  a  fatal  septi- 
caemia in  the  goose.  He,  also,  tried  to  cultivate  it,  but  in  vain.  His 
successors  have  not  been  more  fortunate  in  this  respect.  Here,  then, 

[170]  are  two  micro-organisms,  against  which  natural  immunity  should  be 
easily  obtainable  and  in  a  fashion  quite  other  than  that  against 
anthrax.  Nothing,  indeed,  is  more  frequent  than  examples  of  very 
stable  natural  immunity  against  the  spirilla  of  Obermeyer  and  of 
Sakharoff.  As  I  wished  to  obtain  a  clear  idea  of  the  mechanism  by 
which  the  guinea-pig  resists  injections  of  the  spirillum  of  goose 

[171]  septicaemia  (Spirochaete  anserina)  I  made  injections  of  goose's 
blood,  containing  a  quantity  of  these  organisms,  into  the  peritoneal 
cavity  of  guinea-pigs.  This  injection,  as  usual,  causes  the  disap- 
pearance of  most  of  the  leucocytes,  as  the  result  of  a  very  marked 
phagolysis.  We  know  that,  under  these  conditions,  the  damaged 
leucocytes  allow  a  certain  quantity  of  the  bactericidal  cytase  to 
escape.  In  spite  of  this  the  spirilla  remain  intact  and  exhibit  very 
active  movements  in  the  peritoneal  exudation.  This  exudation,  after 
a  period  of  phagolysis,  which  lasts  for  two  or  three  hours,  begins  to 
be  stocked  again  with  leucocytes  which  come  up  in  increasing 
numbers,  a  fact  that  does  not  prevent  the  spirilla  moving  about  with 
great  rapidity.  Even  seven  hours  after  the  injection  of  goose's  blood 
we  still  find  many  extremely  active  spirilla  among  a  large  number  of 
recently  migrated  leucocytes,  some  of  which  even  at  this  stage  contain 
red  corpuscles  of  the  blood  of  the  goose.  It  is  not  until  later  that 
the  ingestion  of  these  spirilla  by  the  leucocytes  commences,  the 
leucocytes  at  last  damaging  and  completely  destroying  them.  This 
act  of  phagocytosis  may  be  readily  observed  in  hanging  drops  of  the 
peritoneal  exudation  of  inoculated  guinea-pigs.  The  attention  of  the 

1  Ann.  de  Vlnst.  Pasteur,  Paris,  1891,  t.  v,  p.  564. 


Immunity  against  pathogenic  micro-organisms    161 

observer  is  drawn  to  certain  macrophage  leucocytes  which  throw  out 
one  or  two  conical-looking  processes  (Figs.  26—28).  These  pseudopodia 


FIG.  26. — Leucocyte  of 
guinea-pig  in  the  act  of 
ingesting  two  spirilla. 


FIG.  27.— The  same  leuco- 
cyte, half  an  hour  later. 


FIG.  28. — The  same  leuco- 
cyte, ten  minutes  later 
than  Fig.  27. 


attach  themselves  to  spirilla  which  exhibit  very  violent  movements 
as  though  wishing  to  extricate  themselves  from  the  grasp  of  the 
leucocyte.  Sometimes  the  spirillum  succeeds  in  escaping,  but  usually 


FIG.  29. — Leucocyte 
of  guinea-pig  in 
the  act  of  ingest- 
ing a  very  active 
spirillum. 


FIG.  30.— The  same 
leucocyte,  forty 
minutes  later. 


FIG.  31. — The  same 
leucocyte,  half  an 
hour  later  than 
Fig.  30. 


it  becomes  surrounded  by  the  protoplasm  and  sinks  more  and  more 
deeply  into  the  substance  of  the  leucocyte.  Even  when  almost 
surrounded  the  free  part  of  the  spirillum  still  continues  to  move 
(Figs.  29 — 31).  These  movements  cease  only  after  the  complete 
B.  11 


162 


Chapter  VI 


ingestion  of  the  spirillum.    Once  inside  the  phagocyte  the  spirillum 
is  digested  and  soon  becomes  unrecognisable. 

Recently,  Sawtchenko1  took  advantage  of  an  epidemic  of  recurrent 
fever  at  Kazan  to  make  similar  investigations  on  the  natural  im- 
munity of  the  guinea-pig  against  Obermeyer's  spirillum.  He  observed 
that  these  organisms,  when  injected  into  the  peritoneal  cavity, 
remained  there,  alive,  for  24  arid  even  30  hours,  whilst  these  same 
spirilla,  when  kept  at  37°  C.  outside  the  organism  in  their  natural 
medium,  died  at  the  end  of  some  (4 — 7)  hours.  The  injection  of 
[172]  human  serum  containing  spirilla  into  the  peritoneal  cavity  of  guinea- 
pigs  set  up  a  phagolysis  succeeded  by  a  considerable  afflux  of 
leucocytes.  In  spite,  however,  of  the  arrival  of  quite  an  army  of 
these  cells,  the  spirilla  continued  to  move  rapidly ;  for  a  long  time 
they  evaded  the  phagocytes  which,  however,  in  the  end  always 
ingested  them.  But  it  is  only  the  macrophages  which  fulfil  their 
phagocytic  function  (Figs.  32  and  33) ;  the  microphages  obstinately 


Fio.  32. — Macrophage  of  guinea- 
pig  filled  with  spirilla  of  recur- 
rent fever  (after  Sawtchenko). 


Fio.  33. — Macrophage  of  guinea- 
pig  containing  three  Spirochaete 
obermeyeri  (after  Sawtchenko). 


exhibit  an  absolutely  negative  chemiotaxis.  Now,  as  the  macro- 
phages do  not  make  their  way  into  the  peritoneal  cavity  until  after 
the  microphages  have  appeared,  it  is  easy  to  understand  that  phago- 
cytosis can  only  take  place  at  a  late  period.  Sawtchenko  came  to 
the  conclusion  that  "in  the  peritoneal  cavity  of  animals  naturally 
refractory,  the  spirochaetes  perish  as  the  result  of  a  slow  phagocytosis 
and  not  from  the  action  of  the  bactericidal  substances  of  the  fluids." 


1  Arch,  russes  de  pathol.  etc.,  St  Petersb.,  1900,  t.  ix,  p.  578;  and  Sawtchenko  et 
•Melkich,  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  502. 


Immunity  against  patJiogenic  micro-organisms     163 

In  conformity  with  this  result  this  observer  has  often  noted  the 
ingestion  of  living  spirilla  by  the  macrophages,  in  hanging  drops  of 
the  peritoneal  exudation  of  inoculated  guinea-pigs.  The  phenomenon 
corresponds  exactly  to  that  described  in  connection  with  the  spirillum 
of  the  goose. 

In  spite  of  the  great  difference  between  the  spirillum  and  the 
anthrax  bacillus  from  the  point  of  view  of  their  adaptation  to 
surrounding  media,  the  general  result  is  the  same  with  both  these 
microbes  :  animals  endowed  with  natural  immunity  get  rid  of  them 
through  the  agency  of  their  phagocytes. 

It  would  be  impossible  and  even  useless  here  to  pass  in  review  [173] 
all  the  cases  of  natural  immunity  against  infective  micro-organisms. 
We  must  consequently  limit  ourselves  to  several  examples  which 
may  have  an  interesting  bearing  on  the  study  of  the  problem  as 
a  whole.  The  spirilla,  whose  history  we  have  just  recorded,  remain 
in  the  peritoneal  fluid,  without  change  of  form,  up  to  the  moment 
when  they  are  captured  by  the  macrophages.  Let  us  see  by  what 
mechanism  the  natural  immunity  against  micro-organisms,  character- 
ised by  a  very  special  sensitiveness  to  external  influences  and  by  a 
considerable  change  of  shape,  is  produced.  The  cholera  vibrio  and  its 
allies  best  satisfy  this  postulate.  When  they  find  themselves  placed 
under  unfavourable  conditions,  these  vibrios  immediately  become 
transformed  into  small  spherical  bodies  which  are  much  more  like 
cocci  than  vibrios.  The  cholera  vibrio  is  pathogenic  for  the  la- 
boratory rodents,  especially  for  the  guinea-pig,  when  a  fairly  large 
quantity  of  a  culture  is  injected  into  the  peritoneal  cavity.  Against 
smaller  doses,  however,  the  natural  immunity  is  a  most  marked  one. 
If  we  take  a  race  of  the  cholera  vibrio  of  medium  virulence,  and 
inject  into  the  peritoneal  cavity  of  guinea-pigs  a  sublethal  dose  of  a 
culture,  the  following  phenomena  may  be  observed1.  The  inoculated 
vibrios  move  actively  in  the  peritoneal  fluid,  from  which  almost  all 
the  leucocytes  have  disappeared.  There  remain  only  a  few  lympho- 
cytes which  appear  to  be  indifferent  to  the  influences  that  set  up  a 
real  phagolysis.  But,  little  by  little,  fresh  leucocytes  come  into  the 
exudation  and  engage  in  a  struggle  with  the  vibrios  which,  so  long  as 
they  are  free,  retain  their  curved  form  and  complete  motility.  The 
microphages,  especially,  swarm  into  the  peritoneal  cavity.  Some  of 
them  begin  to  ingest  vibrios,  but  this  phagocytosis  is  at  first  slight. 
Later  it  becomes  much  more  active.  The  microphages  and  macro- 
1  Ann.  de  FInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  448. 

11—2 


164 


Chapter  VI 


phages  seize  vibrios  that  are  evidently  living  and  uninjured,  which, 
sometimes,  may  be  observed  inside  the  vacuoles  of  the  leucocytic 
contents  exhibiting  very  lively  movements.  Once  ingested,  however, 
many  of  the  vibrios  become  transformed  into  round  granules.  This 
change  of  shape  is  constant  when  inside  microphages,  but  is  com- 
pletely absent  when  inside  macrophages  (Figs.  34  and  35).  Finally, 


FIG.  34. — Microphage  of  guinea- 
pig  filled  with  cholera  vibrios, 
the  majority  of  which  are 
transformed  into  granules. 


FIG.  35. — Macrophage  of  guinea- 
pig  filled  with  cholera  vibrios 
not  transformed  into  granules. 


[174]  the  phagocytosis  becomes  complete,  and  the  organism  gets  rid  of  the 
vibrios  solely  by  means  of  this  reaction.  Even  seven  hours  after 
injection  of  the  vibrios,  when  the  peritoneal  fluid,  crammed  with 
leucocytes,  has  become  thick  and  turbid,  there  still  remain  a  few 
scattered  vibrios  which  always  retain  their  shape  and  their  normal 
activity.  A  drop  of  this  exudation,  maintained  at  38°  C.  outside  the 
organism,  gives,  in  a  few  hours,  an  abundant  culture  of  very  active 
vibrios.  It  must,  therefore,  be  concluded  that  the  fluid  part  of  the 
exudation  was  powerless  to  destroy  the  vibrios  or  even  to  render 
them  motionless,  whilst  the  living  leucocytes  have  shown  themselves 
capable  of  ingesting  and  digesting  them.  The  peritoneal  exudation, 
withdrawn  at  a  period  when  it  no  longer  contains  any  free  vibrios, 
still  gives  cultures  of  the  organism  for  some  time.  Soon,  however, 
there  comes  a  period  when  the  inoculated  exudation  remains  sterile, 
this  proving  that  the  vibrios,  ingested  in  a  living  state  by  the 
phagocytes,  have  at  length  been  killed  by  the  microphages  and 
macrophages. 


Immunity  against  pathogenic  micro-organisms     165 

When,  instead  of  cholera  vibrios  of  medium  virulence,  we  take 
a  variety  completely  deprived  of  pathogenic  activity,  it  is  sometimes 
observed  that  certain  of  these  organisms,  when  injected  into  the 
peritoneal  cavity  of  the  normal  guinea-pig,  become  transformed  into 
spherical  granules  in  the  fluid  of  the  exudation  without  any  direct  [175] 
co-operation  of  the  phagocytes.  This  transformation  into  granules 
was  first  studied  by  R.  Pfeiffer1  and  hence  has  been  termed  Pfeiffer's 
phenomenon.  It  is  of  limited  occurrence  in  natural  immunity  and  is 
produced,  as  I  have  been  able  to  demonstrate,  only  under  certain 
well  defined  conditions.  Pfeiffer's  phenomenon  is  observed  in  the 
peritoneal  fluid.  It  commences  soon  after  the  injection  of  the  vibrios 
and  takes  place  during  the  period  of  phagolysis.  In  other  parts  of  the 
body  of  the  guinea-pig,  notably  in  the  subcutaneous  tissue  and  in  the 
anterior  chamber  of  the  eye,  Pfeiffer's  phenomenon  does  not  manifest 
itself;  the  animal,  none  the  less,  resists  the  inoculation  of  the  vibrios. 
Even  in  the  peritoneal  cavity,  moreover,  it  is  easy  to  check  the 
granular  transformation  of  the  vibrios  by  means  which  prevent  the 
production  of  phagolysis.  When  we  inject  into  the  peritoneal  cavity 
of  a  guinea-pig  a  foreign  fluid,  capable  of  exciting  the  phagocytic 
action,  e.g.  veal  broth,  physiological  salt  solution,  urine,  etc.,  we  first 
excite  a  transitory  phagolysis.  To  this  stage  succeeds  another  in 
which  the  leucocytes  become  very  numerous  and  much  more  resistant 
than  before.  If  we  take  advantage  of  this  period  of  leucocytic 
stimulation  to  inject  vibrios  which  have  been  attenuated  as  much  as 
possible,  we  shall  observe  that  they  soon  become  the  prey  of  the 
peritoneal  phagocytes,  without  manifesting  any  sign  whatever  of 
Pfeiffer's  phenomenon. 

It  is  evident,  then,  that  this  extracellular  destruction  of  the 
vibrios,  sometimes  observed  in  the  peritoneal  cavity,  is  really  the  work 
of  the  microcytase  that  has  escaped  from  the  phagocytes  during  their 
period  of  transient  injury. 

Having  analysed  the  mechanism  of  natural  immunity  against 
certain  bacilli,  spirilla  and  vibrios,  it  will  be  interesting  to  determine 
whether  the  same  rules  are  to  be  applied  in  the  case  of  the  cocci. 
Choice  is  not  difficult  since  we  may  equally  well  fix  upon  the  staphylo- 
cocci,  the  pneumococci,  streptococci  or  gonococci.  Should  we  decide 
upon  the  streptococcus  it  is  solely  because  the  natural  immunity 
against  this  micro-organism  has  attracted  the  special  attention  of 

1  Ztschr.f.  Hyg.,  Leipzig,  1894,  Bd.  xvin,  S.  1. 


166 


Chapter  VI 


several  observers.  A  second  advantage  of  the  streptococcus,  however, 
is  the  high  degree  of  natural  immunity  manifested  against  it  by 
[176]  a  laboratory  animal  so  convenient  as  the  guinea-pig.  Dr  Jules 
Bordet1  studied  this  subject  in  my  laboratory.  He  observed  that  the 
injection  of  streptococci  into  the  peritoneal  cavity  sets  up  a  marked 
leucocytosis  which  ends  in  a  complete  destruction  of  the  micro- 
organisms. The  leucocytes  rapidly  ingest  the  great  majority  of  the 
streptococci  and  destroy  them  ;  there  remain  only  a  few  isolated  and 
free  individuals  which  are  protected  by  a  clear  zone  (aureola)  which 
develops  around  them,  but  in  the  end  they  also  become  the  victims 
of  the  voracity  of  the  phagocytes.  When  we  increase  the  dose  of 
streptococci  injected,  phagocytosis  still  goes  on,  but  some  of  the 
streptococci  succeed  in  escaping,  and  we  see  a  new  generation 
produced  which  is  distinguished  by  the  thickness  of  the  protective 
aureola.  In  spite  of  the  afflux  of  a  large  number  of  leucocytes, 
they  no  longer  ingest  the  streptococci  and  generalisation  of  the 
infection  results,  followed  by  the  death  of  the  animal.  Natural 
immunity,  then,  can  be  suppressed  under  certain  definite  conditions. 
Dr  Jules  Bordet2  wished  to  satisfy  himself  whether  the  leucocytes 
failed  to  fulfil  their  phagocytic  function  because  of  the  paralysis  of 
their  movements,  or  as  the  result  of  some  other  weakness.  With 
this  object  he  injected  into  the  peritoneal  cavity  of  guinea-pigs,  at 

the  moment  when  the 
streptococci  begin  to  get 
the  upper  hand  of  the 
leucocytes,  a  definite  quan- 
tity of  a  culture  of  Proteus 
vulgaris.  These  small 
bacilli  in  a  short  time 
become  the  prey  of  phago- 
cytes which,  however,  still 
refuse  to  ingest  strepto- 
cocci (fig.  36).  Thereisthus 
in  the  peritoneal  cavity  a 
kind  of  selective  process 
as  regards  the  ingestion 

of  these  microbes.    The  Proteus  disappears  as  the  result  of  phago- 
cytosis, whilst  the  streptococci  thrive  in  the  fluid  of  the  exudation 

1  Ann.  de  VInst.  Pasteur,  Paris,  1897,  t.  xi,  p.  177. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1896,  t.  x,  p.  104. 


FIG.  36. — Peritoneal  exudation  from  guinea-pig 
showing  free  streptococci  and  microphages 
that  have  ingested  Proteus  bacilli. 


Immunity  against  pathogenic,  micro-organisms    167 

and  continue  to  multiply.  This  experiment,  which  readily  succeeds, 
demonstrates  very  clearly  the  difference  between  the  positive  sus-  [177] 
ceptibility  of  the  leucocytes  (with  respect  to  the  Proteus)  and  the 
negative  (with  respect  to  the  streptococcus).  Bordet,  in  accordance 
with  the  view  now  generally  accepted,  regards  this  sensitiveness  as  a 
chemiotaxis,  that  is  to  say  a  perception  of  the  chemical  composition 
of  the  surrounding  medium.  It  must  be  admitted  that  the  substance 
which  excites  the  chemiotaxis  of  the  leucocytes  does  not  readily 
diffuse  and  may  not,  therefore,  be  found  in  a  state  of  solution  in  the 
plasma  of  the  peritoneal  exudation.  Otherwise  the  leucocytes  would 
refuse  to  ingest,  not  only  the  streptococci,  but  also  the  small  Proteus 
bacilli,  bathed  in  the  same  repellent  fluid.  It  is  more  probable  that 
the  substance  which  excites  the  negative  chemiotaxis  is  contained  in 
the  aureola  that  surrounds  the  streptococci,  from  which  it  only 
escapes  with  difficulty  and  for  a  short  distance. 

Marchand1  continued  the  investigation  of  the  same  subject  in 
Denys'  laboratory  at  Louvain.  He  studied  the  natural  resistance  of 
the  guinea-pig,  rabbit  and  dog  against  the  streptococcus.  He,  also, 
came  to  the  conclusion  that  phagocytosis  constitutes  the  principal 
means  of  defence  of  these  mammals  in  their  struggle  against  one  of 
the  most  formidable  of  the  pathogenic  micro-organisms.  Starting 
from  a  single  colony,  Marchand  obtained  two  distinct  races,  one  very 
virulent  for  the  rabbit,  the  other  encountering  a  most  effective 
natural  resistance.  This  resistance  is  due  to  the  activity  of  the 
phagocytes  which  destroy  the  streptococci  in  the  ordinary  fashion. 
He  states  as  the  general  result  of  his  investigation  that  "an  at- 
tenuated streptococcus  is  a  streptococcus  readily  devoured  by  phago- 
cytes "  whilst  "  a  very  virulent  streptococcus  is  a  microbe  that  is  not 
attacked  by  the  leucocytes,"  and  he  adds  that  "a  streptococcus  is 
virulent  because  it  is  not  devoured  by  phagocytes  "  (I.e.  p.  270).  Up 
to  this  point  the  views  of  Marchand  are  in  accord  with  those  of 
Bordet ;  but  here  they  diverge,  in  fact  as  soon  as  it  becomes  a 
question  of  the  explanation  of  the  origin  of  the  difference  in  the 
behaviour  of  the  leucocytes.  Marchand  refuses  to  apply  the  theory 
of  chemiotaxis  and  asserts  "  that  the  phagocytosis  depends  on  some 
physical  property  of  the  streptococcus  and  is  consequently  depen- 
dent on  the  tactile  functions  of  the  leucocytes"  (p.  292).  The 
experiments  upon  which  he  founds  his  conclusion  cannot,  however, 

1  Arch,  de  med.  exper.  et  d'anat.  path.,  Paris,  1898,  t.  x,  p.  253. 


168  Chapter  VI 

be  regarded  as  absolutely  demonstrative.  Thus,  Marchand  observed 
[178]  that  the  attenuated  streptococci,  when  conveyed  in  the  culture- 
fluid  of  the  virulent  variety,  are  as  readily  devoured  by  the  phago- 
cytes as  when  they  were  injected  alone.  According  to  him,  there- 
fore, there  was  in  the  culture-fluid  of  the  virulent  streptococcus 
no  soluble  substance  capable  of  exciting  the  negative  chemiotaxis 
of  the  leucocytes.  But  is  it  quite  proved  that  this  substance  must 
necessarily  pass  into  the  filtrate  of  a  virulent  culture  ?  If  it  adheres 
closely  to  the  glairy  aureola,  as  we  have  suggested,  may  it  not  remain 
behind  with  the  bodies  of  the  streptococci,  without  passing  through 
the  filter  in  any  appreciable  amount?  The  question  cannot  be  re- 
garded as  definitely  settled,  but  probability  appears  to  be  on  the  side 
of  the  theory  of  chemiotaxis. 

Marchand  also  investigated  whether  the  immunity  against  the 
attenuated  streptococcus  might  not  be  explained  by  the  bactericidal 
activity  of  the  fluids  of  refractory  animals.  His  results  were  un- 
varying and  definite.  The  blood  serum  of  his  animals  never  ex- 
hibited any  bactericidal  power  against  the  streptococcus,  and  the 
attenuated  race,  like  the  virulent  one,  grew  well  in  the  serums  of 
the  rabbit,  dog  and  guinea-pig. 

More  recently,  Wallgren1  has  taken  up  the  study  of  the  im- 
munity and  susceptibility  of  rabbits  with  respect  to  the  streptococcus. 
His  conclusions  are,  on  the  whole,  in  accord  with  those  of  his  pre- 
decessors. He  found  that  if  the  injected  streptococci  were  not  very 
virulent  phagocytosis  began  immediately  after  the  injection  into  the 
peritoneal  cavity  and  continued  as  long  as  there  were  any  strepto- 
cocci to  be  attacked.  In  those  cases,  on  the  other  hand,  where  the 
streptococcus  was  endowed  with  a  greater  virulence,  a  transitory 
phagocytosis  took  place  at  the  beginning  of  the  infection ;  but  the 
streptococci  soon  succeeded  in  adapting  themselves  to  the  struggle 
with  the  leucocytes  and  kept  them  at  a  distance.  The  multipli- 
cation of  the  streptococci  could  then  go  on  without  restraint  and 
the  animal  soon  succumbed  to  a  generalised  infection.  Wallgren 
considers  that,  in  the  defence  of  the  organism  against  the  strepto- 
coccus, the  products  of  the  destroyed  leucocytes  may,  sometimes, 
play  a  part. 

As  the  mechanism  of  natural  immunity  against  the  groups  of 
bacteria — bacilli,  spirilla  (and  vibrios)  and  cocci — presents  a  very  great 
analogy  in  all  three,  it  might  be  considered  superfluous  to  continue 
1  Ziegler's  Beitr.  z.path.  Anat.,  Jena,  1899,  Bd.  xxv,  S.  206. 


Immunity  against  pathogenic  micro-organisms    169 


our  analysis  of  this  phenomenon.     Our  review,  however,  would  be 
incomplete  if  we  omitted  to  take  note  of  the  natural  immunity  of  the  [179] 
animal   organism  against  micro-organisms  which  are  distinguished 
by  an  exceptional  toxicity.      The  first  place  in  this  group  must 
undoubtedly  be  assigned  to  the  bacillus  of  tetanus. 

It  may  appear  very  inconsequent  to  be  told  that  animals  very 
susceptible  to  tetanus,  such  as  the  guinea-pig  and  rabbit,  are  endowed 
with  a  natural  immunity  against  the  tetanus  bacillus.  And  yet  this 
fact,  paradoxical  as  it  may  seem,  has  been  demonstrated  beyond 
doubt  by  Yaillard  and  his  collaborators  Vincent  and  Rouget1.  When 
a  small  quantity  of  a  culture  of  the  tetanus  bacillus  was  injected 
into  one  of  the  animals  just  mentioned,  tetanus  was  not  long  in 
declaring  itself.  After  a  period  of  incubation,  certain  muscles  be- 
came stiff  and  a  tetanus,  local  at  first,  soon  became  general  and  had 
a  fatal  issue.  Xow,  when  much  larger  quantities  of  bacilli  are 
inoculated,  but  care  is  taken  to  rid  them  of  the  tetanus  poison  elabo- 
rated in  the  culture-fluid,  the  animals  resist  without  exhibiting  any 
trace  of  tetanus.  This  experiment,  repeated  many  times,  always 
with  the  same  result,  demonstrates  that  the  tetanus  bacillus,  when 
deprived  of  the  co-operation  of 
the  toxin,  encounters,  in  these 
animals  so  susceptible  to  the 
latter,  a  most  effective  opposi- 
tion. Why  is  this?  It  was 
supposed  that,  in  diseases  like 
tetanus  so  markedly  toxic  in 
character,  the  resistance  was 
in  no  way  dependent  on  the 
phagocytic  function.  Thus 
Vaillard  and  Vincent  were 
quite  prepared  to  attribute  no 
share  to  the  phagocytes  in  the 
example  of  natural  immunity 
which  they  had  discovered.  A 
detailed  analysis  of  the  facts 
convinced  them,  however,  that 
in  this  they  were  in  error. 

Guinea-pigs  and  rabbits  do  not  contract  tetanus,  after  the  inoculation 
of  a  quantity  of  spores  and  bacilli  of  tetanus  deprived  of  their  toxin,  [180] 
1  Ann.  de  VInst.  Pasteur,  Paris,  1891,  t.  v,  p.  1 ;  1892,  t.  vi,  p.  385;  1893,  t.  vn,  p.  755. 


FIG.  37.— Leucocytes  of  rabbits  filled 
with  tetanus  spores. 


170  Chapter  VI 

solely  because  of  the  occurrence  of  very  pronounced  phagocytosis. 
Such  an  injection  is  soon  followed  by  a  very  marked  invasion  of 
leucocytes  which  cram  themselves  with  spores  and  bacilli  without 
being  in  any  way  inconvenienced  thereby  (Fig.  37).  Once  the  phago- 
cytes have  devoured  all  these  organisms,  the  latter  become  incapable 
of  producing  their  morbific  effect.  The  spores  cannot  germinate 
within  the  phagocytes,  but  there  undergo  a  marked  degeneration 
and  finally,  after  a  longer  or  shorter  interval,  disappear. 

When,  on  the  other  hand,  the  tetanus  bacilli  or  their  spores  are 
accompanied  by  the  pre-formed  toxin,  the  latter,  according  to  Vaillard, 
excites  a  negative  chemiotaxis  of  the  leucocytes  which  keep  away 
from  the  organisms  and  which  are  thus  allowed  to  multiply  and 
to  secrete  fresh  quantities  of  toxin.  The  natural  immunity  of  the 
animal's  organism  against  the  tetanus  bacillus  can  be  suppressed 
whenever  the  phagocytic  defence  is  hampered  in  any  way.  Under 
natural  conditions  it  is  usually  the  adjuvant  micro-organisms  that  aid 
the  tetanus  infection  by  hindering  the  phagocytes  from  seizing  the 
spores  with  sufficient  rapidity  to  prevent  their  germination.  This 
fundamental  result,  established  by  Vaillard  and  Vincent,  has  often 
been  gainsaid  on  the  evidence  of  insufficient  experiments  (Sanchez- 
Toledo,  Klipstein,  Roncali),  but,  ultimately,  its  accuracy  has  been 
completely  confirmed.  Cases  have  been  cited  in  which  the  tetanus 
spores,  deprived  of  their  toxin,  still  set  up  a  fatal  tetanus.  When  a 
small  fragment  of  an  agar  culture  of  tetanus,  previously  heated  to 
85°  C.  for  the  purpose  of  destroying  the  toxin,  is  inoculated,  we 
produce  tetanus.  Vaillard  and  Rouget  have  demonstrated  that,  under 
these  conditions,  the  leucocytes  penetrate  merely  into  the  superficial 
layer  of  the  agar,  the  spores  germinating  and  the  bacilli  multiplying 
in  the  deeper  part.  We  can  also  set  up  a  fatal  tetanus  in  animals 
by  inoculating,  along  with  sterilised  earth,  spores  deprived  of  their 
toxin  by  means  of  heat.  The  particles  of  soil  protect  the  spores 
against  the  aggression  of  the  phagocytes,  allow  them  to  germinate 
and  then  to  poison  the  organism.  Lactic  acid  produces  an  analogous 
effect,  by  destroying  or  weakening  the  phagocytes.  Micro-organisms, 
most  often  inoffensive  in  themselves,  also  prevent  the  phagocytosis 
of  the  tetanus  spores  and  thus  aid  the  intoxication. 
[181]  The  facts  above  summarised  have  been  demonstrated  to  be  the 
rule  for  several  species  of  anaerobic  pathogenic  bacteria.  Thus, 
Besson1  showed  that  the  septic  vibrio  is,  by  itself,  incapable  of  setting 
1  Ann.  de  Vlnst.  Pasteur,  Paris,  1895,  t.  ix,  p.  179. 


Immunity  against  pathogenic  micro-organisms    171 

up  septicaemia ;  in  order  to  do  this  it  needs  the  co-operation  of  other 
micro-organisms.     Leclainche  and  Vallee1   have  extended  the  same 
rule  to  the  bacillus  of  symptomatic  anthrax  (Bacillus  chauvaei),  so 
important  as  being  the  cause  of  an  epizootic  disease  of  the  Bovidae. 
The  spores  of  this  bacillus  when  heated  to  80° — 85°  C.  lose  the  pre- 
formed toxin  and  at  once  become  incapable  of  producing  infection. 
In  this  case  also,  these  spores  soon  after  injection  become  the  prey 
of  phagocytes,  which  seize  them,  prevent  their  germination  and  check 
their  pathogenic  action.     If  to  these  heated  spores,  however,  we  add 
a  small  quantity  of  toxin,  they  are  enabled  to  germinate  in  the 
tissues  and  set  up  a  typical  infection.     If  heated  spores  are  mixed 
with  sterile  sand,  and  the  mixture  introduced  into  guinea-pigs,  these 
animals  almost  invariably  acquire  a  fatal  symptomatic  anthrax.     The 
spores  in  the  superficial  part  of  the  sandy  mass  are  readily  devoured 
by  the  phagocytes ;  but  those  which  are  included  within  the  central 
part  of  the  mass,  being  protected  for  some  time  against  these  cells, 
germinate  as  soon   as   they  become  permeated  with  the  fluids   of 
the  animal  organism.    If  we  envelope  the  sand  in  a  paper  sac  the 
protection  against  the  phagocytes  is  still  more  complete  and  allows 
almost  all  the  spores  to  germinate  and  in  every  case  to  set  up  a  fatal 
infection.     Leclainche  and  Vallee  conclude  from  their  experiments 
"  that  we  only  require  to  protect  the  spore  mechanically  in  order  to 
see  an  infection  produced;  here  we  cannot  allege  an  increase  of  its 
virulence,  as  when  we  associate  a  chemical  substance  with  the  virus, 
and  the  exclusive  part  played  by  the  phagocytosis  in  the  protective 
process  stands  out  clearly  "  (p.  221). 

The  history  of  these  three  anaerobic  organisms  clearly  proves  that 
the  natural  immunity  against  them  cannot  be  made  dependent  on 
either  the  bactericidal  power  of  the  fluids,  or  on  any  antitoxic  property, 
or  on  the  incapacity  of  the  micro-organism  to  secrete  its  toxin  in  the 
fluids  of  the  refractory  animal.  The  cause  of  this  immunity  resolves 
itself  into  the  reaction  of  the  phagocytes  which  prevent  the  micro- 
organisms from  producing  their  poisons. 

All  that  has  been  said  on  the  subject  of  the  natural  immunity  of 
the  Vertebrates  has  had  reference  to  cases  of  resistance  against  [182] 
Bacteria.  But  may  not  the  immunity  against  micro-organisms  be- 
longing to  other  groups  depend  on  other  factors  with  which  the 
reader  has  not  yet  been  made  sufficiently  acquainted  ?  Amongst  the 
lower  plants  there  are  Blastomycetes  (Torulae  and  Yeasts)  which  are 
1  Ann.  de  Vlnst.  Pasteur,  Paris,  1900,  t.  xiv,  p.  202. 


172  Chapter  VI 

capable    of    producing    infections,    e.g.    the    disease    amongst    the 
Daphniae. 

Some  observers,  no  doubt,  have  come  to  the  conclusion  that  the 
various  Blastomycetes,  when  introduced  into  a  refractory  organism, 
undergo  complete  destruction  within  a  few  hours  without  any  inter- 
vention of  phagocytosis.  Thus  Jona1  explains  the  disappearance  of 
yeast-cells  injected  into  the  veins  or  peritoneal  cavity  of  the  rabbit  as 
due  to  the  sole  influence  of  the  microbicidal  property  of  the  blood- 
fluid.  Gilkinet2  looks  at  it  from  the  same  point  of  view.  He  in- 
jected beer  yeast  (Saccharomyces  cerevisiae)  into  a  rabbit  and  observed 
that  it  had  disappeared  shortly  afterwards.  The  destruction  of  the 
yeast-cells,  according  to  this  observer,  "is  effected  by  means  of 
plasmatic  juices  "  and  "  is  due  to  a  specific  property  of  the  organic 
fluids"  whose  nature  is  "quite  unknown  as  regards  its  essential 
principle."  Phagocytosis  is  said  to  play  no  part  in  this  phenomenon. 
Let  us  hasten  to  say  that  before  the  publication  of  the  two  works  just 
cited,  a  memoir  by  Schattenfroh3  had  appeared  on  the  same  subject. 
This  observer,  who  carried  out  his  experiments  in  Buchner's  laboratory 
at  Munich,  accurately  observed  and  described  the  destruction  of  in- 
jected yeasts  by  phagocytes,  whilst  his  experiments  on  the  microbicidal 
power  of  the  blood  and  serum  failed.  This  testimony  is  the  more 
important  that  it  emanates  from  a  school  by  whom  the  microbicidal 
power  of  the  "  humours  "  is  regarded  as  the  principal  factor  in  the 
defence  of  the  animal  organism.  The  facts  described  by  Schattenfroh 
are  perfectly  accurate  and  have  been  confirmed  in  my  laboratory  by 
Skchiwan4,  who  did  not  restrict  himself  to  injecting  ordinary  yeasts 
(pink  yeast,  Saccharomyces  pastorianus)  but  inoculated  guinea- 
pigs  with  pathogenic  yeast-cells,  isolated  by  Curtis5  from  a  case  of 
[183]  myxomatous  tumour  in  man.  The  guinea-pig  is  refractory  to  small 
doses  of  this  yeast  but  succumbs  to  injections  of  larger  quantities  : 
Skchiwan  convinced  himself  that  the  ingestion  of  the  non-pathogenic 
yeast-cells  takes  place  with  great  rapidity.  Thus  the  Saccharomyces 
pastorianus,  in  the  peritoneal  cavity  of  the  guinea-pig,  is  ingested 
almost  exclusively  by  microphages  at  the  end  of  two  hours.  Some.* 
(3 — 4)  hours  after  injection,  "  sowings  "  of  the  peritoneal  exudatiouy 

Centralbl.f.  Bacteriol  u.  Parasitenk.,  Jena.  1897,  Bd.  xxi,  S.  147. 
Arch,  de  med.  exper.  et  d'anat.  path.,  Paris,  1897,  t.  ix,  p.  881. 
Arch.f.  Hyg.,  Miinchen  u.  Leipzig,  1896,  Bd.  xxvn,  S.  234. 
Ann.  de  VInst.  Pasteur,  Paris,  1899,  t.  xm,  p.  770. 
Ibid.,  1896,  t.  x,  p.  448. 


Immunity  against  pathogenic  micro-organisms    173 

no  longer  yield  growths.  On  the  other  hand  Curtis'  pathogenic 
yeast-cells  resist  the  action  of  the  phagocytes  for  a  much  longer  time. 
After  a  period  of  phagolysis  in  the  peritoneal  cavity,  the  leucocytes 
that  have  just  arrived  in  large  numbers  begin  to  seize  the  yeast-cells. 
Usually  several  macrophages  fuse  around  the  same  yeast  globule  form- 
ing a  very  characteristic  kind  of  rosette.  Sometimes  the  macrophages 
run  together  to  produce  a  giant  cell,  whose  centre  contains  the 
yeast-cell.  This  latter  defends  itself  against  phagocytosis  by  secreting 
a  fairly  thick  membrane.  The  struggle  between  the  two  living 
elements  is  a  fairly  prolonged  one ;  24  to  48  hours  after  inoculation  all 
the  yeasts  are  surrounded  by  phagocytes,  amongst  which  microphages 
are  exceptional.  But  the  parasites  remain  alive  for  4 — 6  days  after 
their  injection  into  the  peritoneal  cavity,  as  proved  by  the  cultures  that 
are  obtained  from  the  exudation  when  the  fluid  is  "  seeded  "  out.  It 
must  be  concluded,  therefore,  that  the  yeast-cells  were  surrounded  by 
the  phagocytes  whilst  still  presenting  all  the  signs  of  life.  Skchiwan 
was  no  more  successful  than  Schattenfroh  in  demonstrating  any  kind 
of  microbicidal  action  of  the  fluids  on  the  Blastomycetes. 

There  is,  consequently,  no  doubt  whatever  that  the  resistance  of 
the  animal  organism  against  yeasts  follows  the  same  rules  that  hold 
in  the  defence  against  bacteria. 

The  animal  micro-organisms  are  much  rarer  in  infective  diseases 
than  are  the  microphytes;  moreover  the  impossibility  of  obtaining 
cultures  of  them  renders  their  investigation  much  more  difficult. 
Yet  there  exist  facts  that  are  capable  of  affording  us  information  as 
to  the  means  made  use  of  by  the  refractory  organism  against  certain 
parasitic  Protozoa.  Amongst  these  latter  the  Trypanosomae  play  a 
most  important  part.  One  species  of  this  genus  (T.  leivisi)  produces 
an  infective  disease  in  rats,  especially  in  the  grey  rat  (Mus  decu- 
manns  ,  the  blood  of  these  rodents  often  containing  a  very  large 
number  of  them,  whilst  the  small  flagellated  organisms  flourish  well  in 
the  serum  prepared  from  the  blood  of  affected  animals.  Laveran  and  [184] 
-  Mesnil1,  in  their  studies  on  the  Trypanosomae,  injected  defibrinated 
blood  containing  numerous  Trypanosomae  into  the  peritoneal  cavity 
of  guinea-pigs,  which  exhibit  a  natural  immunity  against  this  parasite. 
The  parasites  remained  alive  for  some  days  and  then  disappeared 
completely.  Here  again  it  is  the  phagocytes  of  the  peritoneal  exuda- 
tion which  rid  the  animal  of  the  Trypanosomae  by  ingesting  them. 
Laveran  and  Mesnil  were  able,  by  the  examination  of  hanging  drops 
1  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  673. 


174  Chapter  VI 

of  the  peritoneal  exudation  of  their  guinea-pigs,  to  detect  leucocytes 
in  the  act  of  devouring  Trypanosomae  which  showed,  by  their  active 
movements,  that  they  were  still  alive.  Once  the  parasites  were  com- 
pletely enclosed  within  the  macrophages,  their  final  disappearance 
took  place  with  extraordinary  rapidity. 

In  this  chapter  we  have  attempted  to  place  before  the  reader  a 
complete  series  of  the  phenomena  observed  in  natural  immunity 
in  animals.  We  have  passed  in  review  the  resistance  of  the  animal 
organism  against  the  principal  groups  of  bacteria,  and  we  have  dwelt 
on  certain  of  them  which  are  most  capable  of  adapting  themselves 
to  various  media,  and  on  others  which  present  examples  of  micro- 
organisms more  exacting  and  more  delicate.  We  have  examined  the 
immunity  against  Blastomycetes  and  parasitic  animalcules.  Above 
all,  in  the  lower  animals,  just  as  in  the  Vertebrata  of  all  classes,  we 
have  always  observed  this  general  phenomenon :  phagocy tic  resistance 
as  the  principal  and  constant  factor  in  natural  immunity. 


CHAPTER   YII  [IBS] 

THE  MECHANISM  OF  NATURAL  IMMUNITY  AGAINST 
MICRO-ORGANISMS 

The  destruction  of  micro-organisms  in  natural  immunity  is  an  act  of  resorption. — 
Part  played  by  inflammation  in  natural  immunity. — Importance  of  microphagcs 
in  immunity  against  micro-organisms. — Chemiotaxis  of  leucocytes  and  ingestion 
of  micro-organisms. — Phagocytes  are  capable  of  ingesting  living  and  virulent 
micro-organisms. — The  digestion  of  micro-organisms  in  phagocytes  is  most 
often  effected  in  a  feebly  acid  medium. — Bactericidal  property  of  serums. — • 
Phagocytic  origin  of  the  bactericidal  substance. — Theory  of  the  secretion  of  the 
bactericidal  substance  by  leucocytes. — Comparison  of  the  bactericidal  power  of 
serums  and  of  blood  plasmas. — The  bactericidal  substance  of  blood  serums  must 
not  be  considered  a  secretion-product  of  leucocytes;  it  remains  within  the 
phagocytes,  so  long  as  they  are  intact. — The  cytases. — Two  kinds  of  cytases: 
macrocytase  and  microcytase. — Cytases  are  endo-enzymes,  allied  to  trypsins. — 
Changes  in  the  staining  properties  and  in  the  form  of  micro-organisms  in  the 
phagocytes. — Absence  or  rarity  of  fixatives  in  the  serums  of  animals  endowed 
with  natural  immunity. — The  agglutination  of  micro-organisms  does  not  play 
any  important  part  in  the  mechanism  of  natural  immunity. — Absence  of  anti- 
toxic property  of  the  body  fluids  in  natural  immunity. — The  phagocytes  destroy 
the  micro-organisms  without  their  ingestiou  being  preceded  by  neutralisation 
of  the  toxins. 

THE  facts  we  have  set  forth  in  the  preceding  chapter  clearly  justify 
us  in  concluding  that  the  destruction  of  the  micro-organisms  in 
natural  immunity  is  reduced  to  their  resorption  by  the  phagocytes. 

We  have  now,  therefore,  returned  to  the  point  arrived  at  and 
already  studied  in  Chapter  iv,  where  we  attempted  to  establish  certain 
fundamental  laws.  It  remains  to  be  seen  up  to  what  point  these 
laws  apply  to  the  phenomena  of  natural  immunity  against  infective 
micro-organisms. 

The  introduction  into  the  animal  organism  of  foreign  blood,  of 
spermatozoa  belonging  to  the  same  or  a  different  species,  or  of  any 
other  cells,  as  in  the  case  of  the  penetration  of  micro-organisms  into 


176  Chapter   VII 

the  tissues  or  cavities  of  the  body  of  a  refractory  animal,  determines, 
primarily,  a  localised  inflammation,  associated  with  which  is  a  dia- 
pedesis  of  many  white  corpuscles.  Instead  of  aseptic  inflammation,  as 
[186]  in  the  case  of  the  resorption  of  cells,  there  is  produced,  in  antimicrobial 
immunity,  a  septic  inflammation  at  the  point  of  invasion  of  the  micro- 
organisms. In  this  inflammation  the  redness  and  heat  are  slight,  the 
fluid  part  of  the  exudation  is  insignificant,  but  what  is  especially 
characteristic  is  the  large  number  of  leucocytes  which  come  up 
towards  the  point  menaced.  This  constancy  of  the  inflammatory 
reaction  in  natural  immunity  is  one  of  the  best  proofs  of  the  accuracy 
of  the  view  that  inflammation  is  a  phenomenon  useful  to  the  animal 
organism,  especially  in  its  struggle  against  microbial  invasion.  As  we 
have  devoted  a  whole  volume  to  the  discussion  of  the  comparative 
pathology  of  inflammation  it  is  here  unnecessary  to  discuss  it  further. 
Since  the  publication  of  this  book  numerous  articles  on  inflammation 
have  appeared,  but  none  of  them  have  undermined,  in  the  least 
degree,  the  fundamental  bases  of  the  phagocytic  theory  of  inflamma- 
tion. The  view  that  this  phenomenon  really  constitutes  a  healing 
reaction  of  the  organism  is  at  present  accepted  by  many  investigators 
in  all  countries.  It  is  therefore  needless  to  re-defend  it. 

Although  there  still  remain  a  certain  number  of  points  that  are 
not  sufficiently  cleared  up  in  the  essential  mechanism  of  inflammation, 
it  is  now  proved  beyond  doubt  that  the  sensitiveness  of  the  cell 
elements  which  here  play  a  part,  is  one  of  the  essential  factors  in  the 
process.  The  nerve  cells  which  govern  the  vascular  dilatation,  the 
endothelial  cells  which  allow  of  the  passage  of  leucocytes,  and  the 
leucocytes  themselves  which  escape  from  the  vessels  in  order  to  reach 
the  point  of  entrance  of  the  micro-organisms,  all  must  be  influenced 
in  a  special  fashion.  In  natural  immunity  the  phagocytes  exhibit  a 
positive  chemiotaxis  and  this  form  of  sensitiveness  is  a  condition 
indispensable  to  a  state  of  immunity  and  to  the  disappearance  of 
the  micro-organisms. 

In  my  eighth  lecture  on  inflammation  I  have  already  set  forth  the 
fundamental  facts  upon  which  rests  the  doctrine  of  the  chemiotaxis 
of  leucocytes.  During  the  last  ten  years  numerous  data  corrobo- 
rating these  results,  obtained  first  by  Leber,  Massart,  and  Charles 
Bordet,  and  since  confirmed  by  numerous  other  observers,  have  been 
accumulated. 

In  the  resorption  of  blood  corpuscles  and  of  animal  cells  in 
general,  it  is  especially  the  macrophages  which  intervene,  but  in 


Mechanism  of  immunity  against  micro-organisms    177 

natural  immunity  against  micro-organisms  positive  chemiotaxis  is 
exhibited  by  the  microphages  more  than  by  the  macrophages.  [187] 
When  we  examine  an  inflammatory  exudation  and  find  a  prepon- 
derance of  microphages  we  are  satisfied  that  there  has  been  an 
intervention  of  micro-organisms.  Even  in  the  examples  where  it 
is,  at  first,  principally  the  macrophages  which  destroy  the  micro- 
organisms (as  in  the  case  of  the  resistance  of  the  animal  organism 
against  the  tubercle  bacillus),  there  is  also  a  great  afflux  of  micro- 
phages. The  sensitiveness  of  the  two  chief  categories  of  phagocytes 
often  exhibits  a  marked  difference.  We  need  merely  recall  to  the  reader 
the  example  of  the  spirilla,  ingested  and  destroyed  exclusively  by  the 
macrophages  of  the  guinea-pig,  which  alone  exhibit  the  necessary  posi- 
tive chemiotaxis.  In  many  other  examples  of  natural  immunity  the 
part  played  by  the  macrophages  is  masked  by  that  of  the  microphages. 

In  natural  immunity  the  motile  phagocytes,  having  come  up  to  the 
invaders,  perform  a  second  physiological  function  ;  they  ingest  the 
micro-organisms.  Sometimes  the  leucocytes  devour  at  one  swoop 
whole  masses  of  these  organisms,  and  carry  out  their  work  in  a  very 
short  time.  In  other  cases,  especially  when  actively  motile  micro- 
organisms, such  as  the  spirilla  of  Obermeyer  or  of  Sacharoff,  have 
to  be  dealt  with,  the  ingestion  takes  place  with  more  difficulty  and 
requires  special  conditions.  Thus,  in  order  to  ingest  a  spirillum, 
the  macrophages  of  the  guinea-pig  throw  out  long  conical  processes. 
Xever  in  the  ingestion  of  micro-organisms  have  I  observed  methods 
comparable  to  that  by  which  the  macrophages  seize  upon  the  red 
corpuscles  of  birds  or  upon  other  animal  cells. 

Some  observers  have  expressed  the  opinion  that  micro-organisms 
make  their  way  into  the  cells  spontaneously  and  do  not  need  to  be 
drawn  in  by  means  of  protoplasmic  processes  thrown  out  by  the 
phagocytes.  It  is  of  course  indisputable  that  certain  micro-organisms 
may  pass  into  the  interior  of  the  cell  independently  of  any  act  of 
phagocytosis.  Such  is  the  case  with  the  malaria  parasite  and  allied 
species  which  make  their  way  into  the  red  blood  corpuscles.  But 
here  we  are  dealing  with  amoeboid  organisms,  quite  capable  of 
perforating  the  wall  of  the  red  blood  corpuscle  by  means  of  their 
own  pseudopodia.  Bacteria,  which  do  not  possess  amoeboid  move- 
ments, are  deprived  of  this  power  of  invasion.  There  are,  however, 
very  rare  cases  in  which  such  penetration  does  take  place.  For[i88] 
example,  Bizzozero1  has  described  spirilla  in  the  stomach  of  the 

1  Arch.f.  mikr,  Anat.,  Bonn,  1893,  Bd.  XLII,  S.  146. 
B.  12 


178  Chapter  VII 

dog;  these  may  be  found  inside  epithelial  cells.  But  here  these 
actively  motile  bacteria  make  their  way  into  the  interior  of  vacuoles 
which  open  on  the  free  surface.  Attracted,  probably,  by  the  epi- 
thelial secretions  the  spirilla  first  draw  near  to  the  cells  and  then 
take  advantage  of  small  openings  through  which  they  pass  into  the 
secretory  vacuole.  In  almost  all  cases,  however,  living  and  even 
actively  motile  bacteria  are  incapable  of  penetrating  into  cells.  Thus, 
when  we  observe  the  spirilla  of  recurrent  fever  or  of  goose  septicaemia 
in  the  neighbourhood  of  leucocytes,  we  often  see  them  exhibit  very 
brisk  corkscrew  movements  on  the  surface  of  these  cells  without  ever 
being  able  to  invade  them.  On  the  other  hand,  when  the  leucocyte 
sends  out  a  process  towards  the  spirillum  ingestion  rapidly  takes 
place.  In  anthrax  exudations,  or  in  the  spleen  of  animals  that  have 
succumbed  to  anthrax,  large  numbers  of  bacilli  may  often  be  ob- 
served in  the  immediate  neighbourhood  of  the  leucocytes  or  of  the 
cells  of  the  splenic  pulp,  without  a  single  bacillus  being  found  within 
these  cells.  Nor  do  we  -ever  see  any  bacteria  (which  develop 
abundantly  in  a  drop  of  exudation  withdrawn  from  the  organism) 
invade  the  dead  leucocytes,  lying  alongside  them.  Whilst  on  the 
other  hand  we  see  the  micro-organisms  swarming  outside  the  neigh- 
bouring leucocytes  and  occupying  the  free  spaces  between  these 
cells. 

Almquist1  has  recently  described  a  method  by  means  of  which 
micro-organisms  can  be  taken  into  the  substance  of  dead  leuco- 
cytes. He  collects  leucocytes  from  mammalian  blood,  mixes  them 
with  bacteria,  and  centrifugalises  the  mixture  for  some  time.  He 
convinced  himself  that  after  a  not  very  prolonged  contact  the  bacteria 
are  found  within  leucocytes.  Here  Almquist  excluded  phagocytosis, 
properly  so-called,  that  is  to  say,  the  ingestion  of  the  bacteria  by  the 
active  movements  of  the  leucocytes;  but  he  does  not  give  sufficient 
proof  that  the  cells,  in  his  experiments,  were  actually  dead.  He  thinks 
that  the  relatively  low  temperature  (below  15°  C.)  excluded  the 
possibility  of  amoeboid  movement  in  the  leucocytes  of  warm-blooded 
[189]  animals.  This  argument,  however,  does  not  accord  with  actual  fact, 
for  it  is  indisputable — and  we  have  often  convinced  ourselves  of  this— 
that  the  leucocytes  of  man  and  warm-blooded  vertebrates  maintained 
at  even  a  lower  temperature  than  15°  C.  are  quite  capable  of  motion 
and  of  ingesting  foreign  bodies.  In  all  cases,  the  data  as  a  whole,  some 

1  Ztschr.f.  Hyg.,  Leipzig,  1899,  Bd.  xxxi,  8.  507.     See  review  by  Podwyssotsky 
in  the  Arch,  misses  de  Path.,  St  Petersb.,  1899,  t.  vin,  p.  257. 


Mechanism  of  immunity  against  micro-organisms    179 

of  which  we  have  cited  above,  leave  no  doubt  that  the  ingestion  of 
micro-organisms  unprovided  with  amoeboid  powers  takes  place  by 
means  of  active  movements  of  the  living  protoplasm  of  the  leucocytes. 
To  dissipate  any  remaining  doubt  on  the  part  of  the  reader  I  need 
only  recall  Bordet's  investigations,  cited  in  the  preceding  chapter,  of  the 
behaviour  of  leucocytes  in  the  peritoneal  cavity  of  guinea-pigs  inocu- 
lated with  streptococci  and  Proteus  bacilli.  The  leucocytes  of  the 
peritoneal  cavity  allow  the  virulent  streptococci  to  develop  freely,  not 
ingesting  a  single  one,  whilst  the  Proteus  bacilli,  injected  later,  are 
quickly  devoured  and  at  the  end  of  a  very  short  tune  are  all  found 
in  the  substance  of  these  same  phagocytes.  This  example,  so  demon- 
strative, of  the  chemiotaxis  (positive  as  regards  Bacillus  proteus  and 
negative  as  regards  the  streptococcus),  is  at  the  same  time  the  best 
proof  of  the  fact  that  the  ingestion  of  the  micro-organisms  is  a  vital, 
physiological  act  and  not  merely  a  simple  phenomenon  of  mechanical 
penetration  of  micro-organisms  into  the  soft  protoplasm  of  the 
leucocytes. 

It  was  formerly  thought  that  leucocytes,  charged  with  micro- 
organisms, provide  the  latter  with  a  good  culture  medium  and  serve 
also  as  vehicles  of  transport  for  them  from  one  place  to  another  in 
the  living  organism.  This  view  has  often  been  affirmed  without  any 
proof  whatever  being  given  of  it.  It  has  now  been  demonstrated 
to  be  erroneous.  The  micro-organisms,  with  some  rare  exceptions, 
find  within  the  leucocytes  a  very  unfavourable  medium.  Usually 
they  perish  there,  or,  in  the  case  of  very  resistant  micro-organisms, 
such  as  the  tubercle  bacilli  in  refractory  animals  or  the  endospores  of 
certain  bacteria,  without  being  actually  destroyed,  they  are  prevented 
from  germinating  and  multiplying. 

Later,  another  view  has  been  advanced  that  phagocytes  are 
capable  of  ingesting  only  those  micro-organisms  that  have  been 
previously  killed  by  some  substance  which  is  found  outside  the 
defensive  cells.  This  view  is  quite  as  erroneous  as  the  one  we  have 
just  analysed.  The  phagocytes  are  perfectly  capable  of  seizing  and 
devouring  living  micro-organisms.  We  have  only  to  recall  on  this  point 
the  facts  cited  in  the  preceding  chapter  on  the  subject  of  living  [190] 
bacteria  ingested  by  the  leucocytes  of  various  animals,  or  the  history 
of  the  very  active  spirilla  which  retain  their  motility  up  to  the  moment 
when  they  become  completely  enclosed  by  the  protoplasmic  processes 
of  the  leucocytes  of  the  guinea-pig.  Observations  in  vitro  have,  as 
already  described  in  the  same  chapter,  afforded  a  demonstration  of  the 

12—2 


180  Chapter  VII 

ingestion  of  living  flagellated  Infusoria  by  the  leucocytes  of  refractory 
animals. 

These  facts,  fairly  numerous  in  themselves,  are  not,  however,  the 
only  ones  that  might  be  cited  in  favour  of  the  fundamental  thesis  that 
phagocytes  possess  all  the  means  for  incorporating  living  micro- 
organisms. In  my  first  works  on  phagocytosis  I  cited  the  example  of 
amoeboid  cells,  in  the  Invertebrata,  containing  motile  bacteria1,  and 
that  of  leucocytes  of  the  frog  charged  with  motile  bacilli2  of  an 
artificial  septicaemia.  Since  then  the  number  of  similar  cases  has 
increased  considerably.  Nothing  is  easier  than  to  observe  the  phago- 
cytosis of  living  micro-organisms  in  vitro.  Take  a  drop  of  frog's  lymph 
and  add  to  it  a  few  of  the  Bacilli  pyocyanei,  we  soon  observe  the 
struggle  between  the  leucocytes  and  the  very  motile  bacteria,  and 
inside  the  digestive  vacuoles  bacilli  executing  very  pronounced  and 
active  movements. 

The  same  result  may  be  obtained  by  another  method,  by  which  at 
the  same  time  we  gather  information  as  to  the  virulence  of  the  micro- 
organisms ingested  by  the  phagocytes.  The  view  has  often  been 
expressed  that  phagocytes  seize  only  those  bacteria  that  have  been 
deprived  of  their  virulence  by  a  previous  action  of  the  fluids  of 
the  animal  organism ;  consequently  search  has  been  made  for  some 
attenuating  property  of  these  fluids.  We  have  already  answered  this 
objection  in  the  previous  chapter  by  the  citation  of  cases  in  which 
the  exudations  of  refractory  animals,  containing  only  micro-organisms 
ingested  by  the  phagocytes,  were,  nevertheless,  very  virulent  for  sus- 
ceptible animals.  This  question  has  been  especially  discussed  in 
relation  to  the  anthrax  of  frogs,  on  which  subject  several  investigations 
have  been  carried  out,  the  result  of  which  is  completely  convincing. 
Bacilli  ingested  by  the  leucocytes  of  these  Batrachians  retain  their 
full  virulence  for  a  long  time.  Exudations  which  contain  only  iiitra- 
[191]  phagocytic  bacilli,  the  majority  of  which  have  already  lost  their  normal 
staining  by  aniline  dyes,  produce  fatal  anthrax  in  susceptible  animals, 
such  as  the  mouse  and  the  guinea-pig.  Mesnil  has  demonstrated 
the  same  fact  by  using  the  exudations  of  fresh-water  fishes  that  are 
refractory  to  anthrax.  The  same  rule  applies  equally  to  the  exuda- 
tions of  dogs  and  fowls  that  have  been  inoculated  with  the  bacillus. 
Long  before  these  experiments  on  anthrax  were  made,  Pasteur3 

1  Arb.  a.  d.  zool.  Inst.  d.  Univ.  Wien,  1883,  torn,  v,  S.  160. 
8  Biol.  Centralbl.,  Erlangen,  1883-4,  Bd.  in,  S.  562. 
3  Compt.  rend.  Acad.  d.  sc.,  Paris,  1880,  t.  xc,  p.  952. 


Mechanism  of  immunity  against  micro-organisms     181 

had  shown  that  the  virus  of  fowl  cholera,  which  in  the  guinea-pig 
sets  up  a  mild  affection  and  gives  rise  to  the  formation  of  abscesses, 
retains  its  virulence  for  a  considerable  time  in  the  pus  of  these  ab- 
scesses. When  he  injected  rabbits  with  a  small  quantity  of  guinea- 
pig's  pus  developed  at  the  point  of  inoculation  of  the  cocco-bacillus 
of  fowl  cholera,  the  animals  succumbed  to  a  generalised  and  rapid 
infection.  The  conviction  has  since  been  arrived  at  that,  in  the 
guinea-pig,  these  micro-organisms  readily  become  the  prey  of  the 
leucocytes  that  are  present  in  the  exudations. 

The  rule,  therefore,  is  general  that  in  animals  endowed  with 
natural  immunity  the  phagocytes  seize  and  ingest  even  living  micro- 
organisms that  have  retained  their  initial  virulence. 

Once  within  the  phagocytes,  the  micro-organisms  are  surrounded 
by  a  clear  fluid,  which  accumulates  in  vacuoles,  or  they  are  lodged 
directly  in  the  protoplasm.  In  both  cases  the  micro-organisms  are 
subjected  to  a  digestive  action  which  usually  dissolves  them  com- 
pletely. It  is  not  always  easy  to  form  an  idea  of  the  conditions  under 
which  the  intracellular  digestion  takes  place.  At  first1  I  used  a  weak 
solution  of  vesuvin  for  the  purpose  of  gaining  some  idea  as  to  the 
condition  of  the  micro-organisms  that  have  been  ingested  by  the  leu- 
cocytes and  demonstrated  that  the  living  bacteria  remain  unstained 
in  this  solution,  whilst  the  dead  bacteria  take  on  a  somewhat  deep 
brown  stain.  Thanks  to  this  reaction  I  was  able  to  furnish  one  of  the 
proofs  of  the  fact  that  in  immunised  animals  ingested  bacteria  are 
killed  inside  the  phagocytes.  The  use  of  Ehrlich's  neutral  red  (Neu- 
tralrotJi)  gives  us  further  valuable  indications.  This  colour,  quite 
innocuous  for  living  elements,  is  an  excellent  indicator  of  acid  or 
alkaline  reaction.  Plato2,  in  Breslau,  has  carried  out  numerous  [192] 
researches  on  the  staining  of  micro-organisms  by  a  weak  aqueous 
solution  (l°/0)of  this  substance.  He  has  shown  that  "free"  micro- 
organisms remain  alive  in  this  solution  without  taking  on  any  tinge 
of  colour.  On  the  other  hand,  the  same  micro-organisms,  when 
ingested  by  the  phagocytes,  are  stained  brownish-red.  Most  of  these 
stained  organisms  no  longer  exhibit  any  sign  of  vitality ;  but  amongst 
those  within  the  phagocytes  are  some  which,  in  spite  of  being  deeply 
stained,  are  certainly  alive.  Plato  insists  on  the  fact  that  ingested 
micro-organisms  remain  stained  as  long  as  the  phagocytes  are 
alive,  for,  shortly  after  the  death  of  these  cells,  decoloration  of  the 

1  Ann.  de  VInst.  Pasteur,  Paris,  1887,  t.  i,  p.  325. 

2  Arch.f.  mikr.  Anat.,  Bonn,  1900,  Bd.  LVI,  S.  868. 


182 


Chapter   VII 


micro-organisms  and  of  the  intracellular  granules  takes  place.  When 
neutral  red  is  added  to  an  exudation  in  which  the  leucocytes  are 
dead,  the  staining  of  the  ingested  micro-organisms — dead  or  living- 
does  not  take  place.  I  have  myself  verified 
these  observations,  and  Himmel1,  who  has 
carried  out  an  elaborate  investigation  on 
this  subject  in  my  laboratory,  has  con- 
firmed them  in  numerous  cases.  In  the 
third  and  fourth  chapters  of  this  work  I 
have  already  brought  forward  arguments 
in  favour  of  the  view  that  the  staining  of 
the  ingested  elements  indicates  a  feebly 
acid  reaction  inside  the  phagocytes.  Some- 
times this  reaction  manifests  itself  in  the 
digestive  vacuoles ;  in  other  cases  it  is  ex- 
hibited only  in  the  micro-organisms  directly 
lodged  in  the  protoplasm  (Fig.  38).  Whilst 
the  phagocyte  is  still  living  the  acid  juice 
which  fills  the  vacuoles  or  permeates  the 
ingested  organisms  does  not  mix  with  the 
protoplasm  which  is  always  alkaline.  But 
shortly  after  the  death  of  the  phagocytes 
this  mixture  is  effected  without  difficulty, 
and  the  alkalinity  of  the  protoplasm  is  then 
amply  sufficient  to  neutralise  or  even  render 
alkaline  the  feebly  acid  juices.  This  in- 
terpretation of  the  facts  is  in  complete 

harmony  with  all  the  data,  collected  up  to  the  present,  on  the  staining 
by  neutral  red  of  phagocytised  micro-organisms. 

All  ingested  bacteria  do  not,  however,  stain  in  the  way  we  have 
indicated.  Tubercle  bacilli,  even  in  cases  of  natural  immunity, 
remain  unstained  inside  the  phagocytes  or  take  on  only  a  very  slight 
straw-yellow  tint.  Himmel  made  this  observation  on  the  bacilli  of 
avian  tuberculosis  that  had  been  ingested  by  the  peritoneal  leuco- 
cytes of  the  guinea-pig,  a  species  resistant  to  this  micro-organism. 
It  might  be  thought  that  such  a  resistant  membrane  as  that  of  the 
tubercle  bacillus,  with  its  waxy  layer,  would  prevent  the  penetration 
of  the  acid  leucocytic  juice;  but  several  bacilli  which  resist  de- 
coloration by  acids,  as  do  the  tubercle  bacilli,  notably  the  bacilli 
1  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  928. 


FIG.  38. — Peritoneal  macro- 
phage  of  guinea-pig  that 
has  ingested  a  number 
[193]  of  Bacilli  coli.  Stained 
intra  vitam  with  neutral 
red. 


Mechanism  of  immunity  against  micro-organisms    183 

of  Moeller  and  their  allies,  are  stained  a  bright  red  by  neutral  red 
as  soon  as  they  are  ingested  by  the  phagocytes.  It  is,  therefore, 
more  probable  that,  in  the  case  of  true  tubercle  bacilli,  the  reaction 
in  the  cells  is  no  longer  acid,  but  alkaline.  This  conclusion  is  con- 
firmed by  what  is  observed  in  the  giant  cells  of  the  Algerian  gerbil 
(Meriones  shawii),  a  species  of  rodent  which  exhibits  a  great  natural 
resistance  against  the  bacillus  of  human  tuberculosis1.  The  bacilli, 
ingested  by  these  phagocytes,  secrete  a  series  of  concentric  membranes 
which  become  impregnated  with  phosphate  of  lime  (Fig.  5).  The 
process  causes  the  death  of  the  bacilli,  of  which  there  remain  only 
the  calcined  membranes.  The  precipitation  of  the  lime  salt  around 
bacillary  membranes  itself  indicates  the  alkaline  reaction  of  the 
medium.  The  use  of  certain  staining  substances  fully  confirms  this 
conclusion.  Thus,  with  alizarin  sulpho-acid  the  giant  cells  stain  deep 
violet,  this  affords  clear  proof  of  a  very  distinct  alkaline  reaction. 

We  arrive  then  at  the  general  conclusion  that  phagocytic  digestion 
usually  takes  place  in  a  medium  weakly  acid,  but  that  it  can  also  go 
on  in  an  alkaline  medium.  It  is  impossible,  in  the  present  state  of 
our  knowledge,  to  define  the  nature  of  the  acid  secreted  by  the 
phagocytes.  H.  Kossel2  has  expressed  the  view  that  the  intracellular 
digestion  of  micro-organisms  is  effected  by  the  nucleic  acid,  secreted  [194] 
by  the  cell  nucleus  and  accumulated  in  the  vacuoles  of  the  contents 
of  the  phagocytes.  He  has  brought  forward  in  support  of  this  view 
the  fact  that  nucleic  acid  is  distinctly  bactericidal,  killing  certain 
pathogenic  micro-organisms,  and  giving  a  precipitate  composed  of 
albumen  and  nucleic  acid.  Later  A.  Kossel  pointed  out  the  presence 
in  these  formed  elements  of  albuminoid  substances  which  have 
an  alkaline  reaction  but  which  also  destroy  bacteria.  Thus  he 
has  isolated  from  the  spermatic  fluid  of  the  sturgeon  a  protamine, 
"Sturin,"  which,  even  in  very  weak  solutions,  exhibits  a  strong 
bactericidal  action  on  the  typhoid  bacillus,  staphylococcus,  etc.  It 
is  possible  that  these  substances  play  a  part  in  intracellular  digestion. 
On  the  other  hand,  however,  we  must  regard  it  as  well  established 
that  in  phagocytes  there  is  a  soluble  ferment  which  kills  and 
digests  micro-organisms.  We  have  already  seen,  in  connection  with 
the  resorption  of  animal  cells,  that  it  is  the  ferment  alexine,  or  cytase, 
which  plays  the  principal  part  in  the  digestive  function.  We  must 

"Le9ous  sur  la  pathologie  comparee  de  I'lnflammatioii,"  Paris,  1892,  p.  193; 
authorised  English  translation,  London,  1893,  p.  162. 
2  Arch.f.  Physiol,  Leipzig,  1894,  S.  200. 


184  Chapter   VII 

now  ask  ourselves  whether  the  same  substance  acts  also  on  micro- 
organisms. 

For  more  than  fifteen  years  a  study  of  the  bactericidal  power  of 
the  blood  and  other  fluids  drawn  from  the  animal  organism  has 
been  carried  on.  Based  on  the  not  very  definite  results  of  Traube 
and  Gscheidlen1,  Fodor2  drew  attention  to  the  property  of  the  de- 
fibrinated  blood  of  the  rabbit  to  destroy  the  bacteria  sown  in  it. 
Under  the  inspiration  of  Fliigge3,  Nuttall4  carried  out  a  whole  series 
of  experiments  on  this  bactericidal  property  of  defibrinated  rabbit's 
blood,  of  the  aqueous  humour,  and  of  some  other  fluids.  After 
confirming  Fodor's  general  result,  Nuttall  went  further  and  showed 
that  the  bactericidal  power  of  the  fluids  is  due  to  a  substance  of 
undetermined  nature  which  is  destroyed  by  heating  to  55°  C.  for 
one  hour.  This  discovery  was  confirmed  by  a  large  number  of 
observers,  and  soon  became  an  accepted  fact. 

Fliigge  now  considered  that  he  could  base  a  theory  of  immunity 
on  the  presence  of  the  bactericidal  substance  of  the  body  fluids. 
Bouchard6  and  his  school  adopted  and  developed  this  view,  especially 
with  reference  to  researches  on  the  microbicidal  power  of  blood 
[195]  serum.  Buchner6  soon  came  forward  as  the  chief  advocate  of  this 
theory,  and  enriched  it  by  numerous  investigations  carried  out  by 
himself  or  along  with  collaborators  in  his  school  at  Munich.  It  is  to 
him  that  we  owe  the  suggestion  of  the  term  alexine  (protective 
substance)  to  designate  the  bactericidal  substance  of  blood  serum 
and  other  fluids  of  the  animal  organism  which  are  capable  of 
killing  micro-organisms.  Buchner  determined  the  conditions  under 
which  alexine  acts  best  as  a  bacterial  poison  and  developed  the 
humoral  theory  of  natural  immunity,  according  to  which  the  latter 
is  reduced  to  the  bactericidal  property  of  the  body  fluids. 

As  the  postulates  of  this  theory  are  often  not  in  accord  with  the 
real  facts,  as  Lubarsch7,  especially,  has  demonstrated  in  many  of  his 

1  Jahresb.  d.  schles.  Gesellsch.f.  vaterl.  Cultur,  Breslau,  1874. 

2  Deutsche  med.  Wchnschr.,  Leipzig,  1886,  S.  617  ;  1887,  S.  745. 

3  Ztschr.f.  Hyg.,  Leipzig,  1888,  Bd.  iv,  S.  208. 

4  Ztschr.f.  Hyg,  Leipzig,  1888,  Bd.  iv,  S.  353. 
"  Les  microbes  pathogenes,"  Paris,  1892. 

6  Arch.f.  Hyg.,  Munchen  u.  Leipzig,  1890,  Bd.  10,  S.  84;  CentralU.f.  Bakteriol. 
u.  Parasitenk.,  Jena,  1889,  Bd.  v,  S.  817,  and  Bd.  vr,  SS.  1,  561;  1890,  Bd.  vin, 
S.  65. 

7  CentralU.f.  Bakteriol.  u.  Parasitenk.,  Jena,  1889,  Bd.  vi,  S.  481;  Ztschr.f. 
klin.  Med.,  Berlin,  1891,  Bde  xvni,  xix. 


Mechanism  of  immunity  against  micro-organisms    185 

papers,  we1  expressed  the  opinion  that  a  portion  at  least  of  the  bacteri- 
cidal power  might  come  from  substances  that  had  escaped  from  the 
leucocytes  during  the  preparation  of  the  defibriuated  blood  and  of 
the  blood  serum.  This  hypothesis  remained  for  several  years  un- 
noticed, but  later  several  observers  have,  quite  independently,  arrived 
at  the  conclusion  that  alexine  is  nothing  but  a  leucocytic  product. 
Denys  and  Havet2  were  the  first  to  show  that  exudations  rich  in 
white  corpuscles  exhibited  a  bactericidal  power  much  higher  than  that 
of  the  corresponding  blood  serums.  Shortly  afterwards  H.  Buchner3 
showed  the  same  thing  on  comparing  the  bactericidal  power  of 
exudations  rich  in  leucocytes  with  the  blood  serum  of  the  same 
animals.  As  this  property  disappeared  from  both  fluids  after  they 
had  been  heated  to  55°  C.,  Buchner  concluded  that  the  bacteri- 
cidal substance  of  the  exudations  must  be  identical  with  the  alexine 
of  the  blood  serum.  Several  other  observers,  amongst  whom  Bail, 
Schattenfroh,  Jacob  and  Lowit,  may  be  cited,  obtained  results  more 
or  less  in  accord  with  the  above,  though  obtained  by  different 
methods,  so  that  it  has  now  for  some  time  come  to  be  recognised 
that  the  leucocytic  origin  of  the  alexines  is  generally  accepted, 
especially  since  Jules  Bordet4,  in  an  investigation  carried  out  in  my  [196] 
laboratory,  arrived  at  the  same  result  from  various  very  demon- 
strative experiments. 

Nevertheless  several  authoritative  voices  have  been  raised  against 
this  interpretation  of  the  facts.  R.  Pfeiffer  especially,  with  his  school, 
has  pronounced  against  the  leucocytic  origin  of  the  bactericidal  sub- 
stance found  in  the  blood  serum.  Pfeiffer  and  Marx5  and  Moxter6 
have  insisted  on  the  fact  that  the  fluids  of  exudations  rich  in  leuco- 
cytes are  often  much  less  bactericidal  than  is  the  serum  of  the  blood 
of  the  same  animals. 

For  some  years,  struck  by  the  marked  difference  between  the 
phagocytic  function  of  the  macrophages  and  that  of  the  microphages, 
I  have  thought  that  the  contradictory  results  of  the  observers  cited 
might  be  explained  by  some  difference  in  the  nature  of  the  leucocytes 
of  the  various  exudations  and  of  the  blood  which  served  for  the 


Ann.  de  llnst.  Pasteur,  Paris,  1889,  t.  m,  p.  670. 

2  La  Cellule,  Lierre  et  Louvain,  1894,  t.  x,  p.  7. 

3  Milnchen.  med.  Wchnschr.,  1894,  S.  717. 

4  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  462. 
6  Ztschr.f.  Hyg.,  Leipzig,  1898,  Bd.  xxvn,  S.  272. 

6  Deutsche  med.  Wchnschr.,  Leipzig,  1899,  S.  687. 


186  Chapter  VII 

preparation  of  the  serums.  I  therefore  asked  Gengou  to  devote  his 
attention  to  this  particular  point  and  to  compare  the  bactericidal 
power  of  exudations,  rich  in  microphages,  with  that  of  others  con- 
taining many  macrophages  and  also  with  the  blood  serum  of  the 
same  animals.  Gengou1  has  carried  out  his  experiments  with  remark- 
able exactness  and  care,  and  as  I  have  followed  them  closely  I  am  in 
a  position  to  speak  as  to  their  extreme  accuracy. 

In  order  to  obtain  exudations  very  rich  in  microphages  Gengou 
injected  gluten-casein  by  Buchner's  method  into  the  pleural  cavity 
of  dogs  and  rabbits.  Usually  at  the  end  of  24  hours  he  was  able  to 
collect  a  large  quantity  of  fluid  containing  numerous  leucocytes,  almost 
exclusively  microphages.  To  obtain  macrophagic  exudations  Gengou 
injected  washed  red  blood  corpuscles  of  the  guinea-pig  into  the  pleural 
cavity  of  his  animals  ;  two  days  afterwards  he  withdrew  from  the 
pleural  cavity  a  very  viscid  fluid,  containing,  as  regards  formed 
elements,  macrophages  almost  exclusively.  After  isolation  of  the 
leucocytes  by  centrifugalisation  of  the  exudations,  Gengou  washed 
the  cells  with  physiological  salt  solution  and  then  added  to  them 
an  equal  volume  of  broth.  This  mixture  was  frozen  by  Buchner's 
method,  and  was  then  submitted  to  a  temperature  of  37°  C.  Under 
[197]  these  conditions  the  leucocytes,  killed  by  cold,  gave  up  to  the  fluid 
their  bactericidal  substance. 

Studied  in  this  way,  the  bactericidal  power  of  the  extract  of  micro- 
phages showed  itself  always  superior  to  that  of  the  corresponding 
blood  serum.  The  greatest  difference  was  observed  in  the  dog, 
where,  as  already  mentioned  in  the  preceding  chapter,  the  serum 
of  the  blood  has  no  bactericidal  property  as  regards  the  anthrax 
bacillus,  whilst  the  extract  of  microphages  manifests  this  property 
very  strongly.  The  microphagic  extract  of  the  exudations  of  rabbits 
was  more  active  in  the  destruction  of  the  bacilli  of  anthrax  and 
typhoid,  Bacillus  coli  and  the  cholera  vibrio,  than  was  the  blood  serum. 

The  result  of  these  experiments  leaves  no  room  for  doubt.  The 
microphages,  collected  in  the  aseptic  exudations  of  the  dog  and 
rabbit,  contain  more  bactericidal  substance  than  does  the  blood 
serum  of  the  same  animals.  Nor  can  there  be  a  doubt  that  this 
bactericidal  substance  is  the  same  whether  it  appears  in  the  micro- 
phages or  in  the  blood  serum  :  in  both  cases  it  is  destroyed  by  heating 
to  55°  C.  and,  in  all  other  respects,  it  behaves  in  the  same  manner. 

1  Ann.  de  Vlnst.  Pasteur,  Paris,  1901,  t.  xv,  p.  68. 


Mechanism  of  immunity  against  micro-organisms    187 

The  experiments  of  Gengou  with  the  extracts  of  macrophages 
have  demonstrated,  on  the  other  hand,  that  this  fluid  exerts  no 
bactericidal  power.  Let  it  be  understood  at  the  outset  that  this 
fact  is  in  no  way  an  indication  of  the  absence  of  the  bactericidal 
ferment  in  the  macrophages.  Direct  examination  of  the  phenomena 
which  are  manifested  inside  these  cells  demonstrates  most  clearly  that 
the  macrophages  kill  and  digest  micro-organisms.  But  this  process 
usually  goes  on  much  more  slowly  in  the  macrophages  than  in  the 
microphages,  owing  probably  in  the  former  to  the  presence  of  a 
smaller  quantity  of  the  bactericidal  substance.  Under  these  conditions 
we  can  readily  understand  that  this  substance  does  not  pass,  or  passes 
only  in  small  amount,  into  the  extracts.  There  is  nothing  remark- 
able in  the  fact  that,  with  so  imperfect  a  method  of  preparing  the 
extracts,  the  greater  part  of  the  bactericidal  substance  should  remain 
in  the  bodies  of  the  cells. 

The  facts  just  set  forth  afford  a  sufficient  explanation  of  the  marked 
difference  in  the  results  obtained  by  various  observers  as  to  the 
bactericidal  power  of  the  exudations.  When  the  latter  are  rich  in 
microphages,  the  bactericidal  property  is  very  marked  :  when,  on 
the  other  hand,  the  exudations  contain  a  large  number  of  macro- 
phages, the  bactericidal  power  may  be  very  weak  or  even  nil. 

The  experiments  above  summarised  confirm  the  conclusion  that  [198] 
the  microphages  must  be  regarded  as  the  source  of  the  bactericidal 
substance  of  the  body  fluids.  But  here  arises  the  question :  Do  the 
microphages  secrete  the  substance  during  life,  giving  it  up  to  the 
blood  plasma,  or  does  this  substance  escape  only  after  the  death  of  the 
leucocytes  and  the  damaging  of  the  cells,  due  to  various  external 
causes?  We  here  touch  on  a  problem  which  has  been  the  subject 
of  much  discussion  and  one  of  very  great  importance  in  connection 
with  the  question  of  Immunity  in  general. 

After  the  discovery  of  the  bactericidal  power  of  serums,  several 
investigators  set  to  work  in  search  of  the  source  of  the  bactericidal 
substance.  Hankin1,  and  shortly  afterwards  Kanthack  and  Hardy2,  ex- 
pressed the  view  that  this  substance  is  the  secretion-product  of  the 
eosinophile  leucocytes  which  would  thus  appear  to  be  a  kind  of  motile 
unicellular  glands.  This  theory  could  not  be  supported  by  solid 

1  Centralblf.  Bakteriol.  u.  Parasitenk.,  Jena,  1892,  Bd.  xii,  SS.  777,  809;  1893, 
Bd.  xiv,  S.  852. 

2  Proc.  Roy.  Soc.  London,  1892,  Vol.  LII,  p.  267  ;  Phil.  Trans.,  London,  1894, 
(B)  Vol.  185,  pt.  i,  p.  279. 


188  Chapter   VII 

arguments  and  must  be  regarded  as  generally  abandoned,  because 
it  is  now  completely  out  of  accord  with  well-established  facts.  Thus, 
various  osseous  fishes,  in  spite  of  the  total  absence  of  eosinophile 
or  pseudo-eosinophile  granules  are  none  the  less  capable,  thanks  to 
their  leucocytes,  of  destroying  a  large  number  of  pathogenic  micro- 
organisms (Mesnil,  I.  c.\ 

A  similar  theory  was  enunciated  by  H.  Buchner1,  though  he  holds 
that  it  is  not  the  eosinophile  leucocytes  only  that  secrete  the  bacteri- 
cidal substance,  but  the  leucocytes  in  general.  Being  attracted  to 
the  point  menaced  by  the  micro-organisms,  these  cells  secrete 
their  bactericidal  product,  which  diffuses  into  and  along  with  the 
plasma  of  the  exudations  and  of  the  blood.  In  these  fluids  the 
micro-organisms  undergo  a  more  or  less  complete  destruction,  or 
at  least  severe  injury  which  renders  them  more  susceptible  to  the 
attack  of  the  phagocytes.  At  the  International  Congress  of  Hygiene, 
held  at  Budapest  in  1894,  Buchner  proclaimed  the  thesis  that  "the 
leucocytes  fulfil  an  important  function  in  the  natural  defence  of  the 
organism... by  means  of  soluble  substances  which  they  secrete." 
Later,  his  pupils,  Hahn2  and  Schattenfroh3,  endeavoured  to  support 
[199]  this  theory  by  exact  experiments,  but  they  found  it  impossible  to  do 
this  at  all  satisfactorily.  Later,  another  of  Buchner's  pupils,  Lascht- 
schenko4,  published  a  paper  in  which  he  maintains  that  he  has  found  a 
convincing  argument.  It  is  as  follows.  A  blood  serum,  by  itself  void 
of  bactericidal  property,  some  minutes  after  white  corpuscles  from 
another  species  of  mammal  have  been  added  to  it  acquires  this  pro- 
perty. Thus  the  rabbit's  leucocytes  added  to  dog's  serum  imme- 
diately give  to  it  the  bactericidal  power,  so  long  as  a  large  number 
of  cells  remain  alive  and  motile.  But  when  the  leucocytes  of  the 
same  species  are  added  to  rabbits'  serum  the  fluid  becomes  no  more 
bactericidal  than  before.  The  same  result  may  be  obtained  by  mixing 
rabbits'  leucocytes  with  the  blood  serum  of  the  horse,  pig  and  other 
species.  Laschtschenko  concludes  from  these  observations  that  the 
vital  secretion  of  the  bactericidal  substance  by  the  leucocytes  of  the 
rabbit  takes  place  when  they  are  irritated  by  the  serum  of  a  different 
species.  As  an  analogous  effect  has  been  observed  with  mixtures  of 

1  Munchen.  med.  Wchnschr.,  1894,  S.  717  and  1897,  S.  1320. 

2  Arch.  f.  Hyg.,  Munchen  u.  Leipzig,  1895,  Bd.  xxv,  S.  105  ;  1897,  Bd.  xxvui, 
S.  312.    Berl  klin.  Wchnschr.,  1896,  S.  864. 

3  Arch.  /.  Hyg.,  Munchen  u.  Leipzig.,  1897,  Bd.  xxxi,  p.  1 ;   1899,  Bd.  xxxv, 
S.  135.    Munchen.  med.  Wchnschr.,  1898,  SS.  353,  1109. 

4  Arch.f.  Hyg.,  Munchen  u.  Leipzig,  1900,  Bd.  xxxvn,  S.  290. 


Mechanism  of  immunity  against  micro-organisms    189 

rabbits'  leucocytes  with  the  serum  of  a  different  species  heated  to 
60°  C.,  Laschtschenko  believes  himself  safe  from  the  objection  that  the 
giving  up  of  the  bactericidal  substance  results  from  the  death  or 
injury  of  the  white  corpuscles.  According  to  him  this  injurious 
effect  on  the  white  corpuscles  can  only  be  produced  by  an  unstable 
substance  which  is  destroyed  by  heating  to  60°  C.  Laschtschenko 
forgets  that  the  leucocytes  are  in  general  delicate  cells,  capable  of 
being  affected  even  by  fluids  which  do  not  actually  kill  them.  Now 
we  know  that  serums,  when  heated  to  60°  C.,  still  retain  their  power 
of  agglutinating  the  leucocytes,  a  power  which  must  hamper  these 
cells  in  their  normal  function. 

Trommsdorff1,  in  an  investigation  carried  out  in  Buchner's  labo- 
ratory, endeavoured  to  supplement  Laschtschenko's  results  and  to 
support  them  by  new  and  more  convincing  experiments.  But  he 
only  succeeded  in  a  few  cases  in  obtaining  a  bactericidal  serum  after 
adding  rabbits'  leucocytes  to  the  blood  serum  of  other  animals. 
"In  a  great  number  of  my  experiments,"  says  Trommsdorff,  "I 
very  often  did  not  succeed  in  extracting  the  alexines  from  the 
rabbit's  leucocytes  by  the  use  of  Laschtschenko's  method"  (p.  385). 
On  the  other  hand,  Trommsdorff,  wishing  to  establish  the  living 
condition  of  the  leucocytes  mixed  with  a  foreign  serum,  arrived  at 
the  following  result:  "In  the  majority  of  the  cases,  as  in  fresh 
exudations,  the  number  of  living  leucocytes  after  their  treat- [200] 
ment  Avith  active  horse's  serum,  as  well  as  with  inactive  serum 
(heated  to  60°  C.)  of  dog,  ox  and  horse,  varied  between  60  and 
80  °/0"  (p.  391).  In  spite  of  these  verifications,  Trommsdorff  comes 
to  the  conclusion  that  the  presence  of  alexine  in  those  serums  to 
which  leucocytes  had  been  added,  must  "  in  all  probability "  be 
attributed  to  its  secretion  by  the  living  leucocytes.  We  regard  it 
as  much  more  probable  that  the  alexine,  in  those  cases  where  it 
passed  into  the  serum,  was  due  to  the  breaking  up  of  the  dead 
leucocytes,  whose  numbers  rose  to  40  °/o>  that  is  to  say,  almost 
half  their  total  number.  Our  conclusion  is,  in  any  case,  much 
more  in  accord  with  the  more  constant  and  more  exact  results 
obtained  by  other  methods. 

In  spite  of  the  insufficiency  of  proofs  in  favour  of  the  theory  of 

bactericidal  secretions  by  the  leucocytes  it  has  been  very  favourably 

received  by  many  investigators.     As,  however,  it  came  into  collision 

with  the  general  fact  that,  in  the  refractory  animal,   the  micro- 

1  Arch.f.  Hyg,,  Miinchen  u.  Leipzig,  1901,  Bd.  XL,  S.  382. 


190  Chapter  VII 

organisms  remain  alive  in  the  plasmas  of  the  exudations  and  are,  in 
this  condition,  ingested  by  the  phagocytes,  it  was  therefore  very  im- 
portant that  this  fundamental  contradiction  should  be  settled  by 
decisive  experiments.  The  attempt  has  often  been  made  to  obtain 
blood  plasma  and  to  compare  its  bactericidal  action  with  that  of 
serum  from  the  same  animal.  In  the  preceding  chapter  we  have  already 
mentioned  an  attempt  in  this  direction  made  by  Sawtchenko.  Hahn1 
had  previously  attempted  to  prepare  plasma  by  adding  histon  to  blood. 
As  this  "  plasma "  was  found  to  be  just  as  bactericidal  as  the  blood 
serum  Hahn  concluded  that  the  bactericidal  substance,  secreted  by 
the  living  leucocytes,  circulates  in  the  living  blood.  In  all  the  experi- 
ments carried  out  by  this  method  it  was  impossible  to  avoid  certain 
sources  of  error,  and  in  my  laboratory  Gengou2  undertook  a  new 
series  of  researches,  endeavouring  to  obtain  from  blood  a  fluid  re- 
sembling normal  plasma  as  closely  as  possible.  The  method  he 
employed  has  been  described  in  detail  in  a  memoir,  on  an  anti- 
coagulating  serum,  which  he  published  along  with  Bordet3.  The 
blood  was  drawn  into  paraffined  tubes  and  centrifugalised  at  once 
in  other  tubes  whose  walls  were  likewise  covered  with  a  layer  of 
[201]  paraffin.  The  fluid  thus  prepared  is  certainly  more  allied  to  circu- 
lating plasma  than  is  the  blood  serum  obtained  after  the  coagulation 
of  the  blood.  Nevertheless,  it  is  still  far  from  being  identical  with 
true  normal  plasma ;  it  still  coagulates,  though  tardily.  Gengou 
compared,  in  their  bactericidal  action,  the  blood  serum  and  the  serum, 
decanted  after  the  tardy  coagulation  of  the  fluid  analogous  to 
plasma.  He  carried  out  a  great  number  of  experiments  with  the 
two  fluids,  obtained  from  dogs,  rabbits  and  rats,  making  a  comparative 
study  of  their  bactericidal  power  as  regards  the  anthrax  bacillus,  the 
typhoid  bacillus,  and  the  cholera  vibrio.  I  have  closely  followed  all 
these  experiments  and  can  confirm  the  results  described  by  Gengou, 
namely,  that  the  fluid,  in  this  plasma  serum,  possesses  an  insignificant 
bactericidal  power  or  none  at  all,  whilst  the  blood  serum  almost 
always  exhibits  this  property  to  a  marked  degree. 

As  a  result  of  the  researches  just  summarised  it  is  no  longer 
possible  to  maintain  the  theory  of  bactericidal  secretions  by  leuco- 
cytes or  by  any  other  category  of  cells.  The  bactericidal  substance 

1  Arch.  f.  Hyg.,  Miinchen  u.  Leipzig,  1895,  Bd.  xxv,  S.  105;  Bert,  kiin.  Wchnschr., 
1896,  S.  864. 

2  Ann.  de  Hnst.  Pasteur,  Paris,  1901,  t.  xv,  p.  232. 

3  Ibid.,  p.  129. 


Mechanism  of  immunity  against  micro-organisms    191 

does  not  circulate  in  the  blood  plasma  nor  in  that  of  the  exudations, 
and  this  is  a  sufficient  reason  for  refusing  to  it  the  title  of  a  secretion- 
product.  Its  presence  in  the  blood  serum  is  due,  like  that  of  the 
fibrin-ferment,  to  the  destruction  or  more  or  less  grave  injury  of 
the  phagocytes. 

This  fact,  upon  which  we  must  insist  most  strongly,  is  in  flat  con- 
tradiction to  the  view  recently  formulated  by  Wassermann1.  In  a  work 
devoted  to  natural  immunity  against  micro-organisms,  this  author 
describes  how  he  submits  his  animals  (guinea-pigs)  to  the  action  of  an 
anticytase  (or  anti-alexine)  serum  whose  preparation,  described  in  the 
fifth  chapter  of  this  work,  offers  no  difficulties.  Under  the  influence  of 
this  serum,  the  guinea-pigs,  into  the  peritoneal  cavity  of  which  a  strong 
dose  of  typhoid  cocco-bacilli  is  inoculated,  die  from  infection,  whilst  the 
control  animals,  inoculated  in  a  similar  manner,  but  which  have  re- 
ceived in  addition  some  normal  rabbit's  serum,  heated  to  60°  C.,  entirely 
resist  the  infection.  Wassermann  concludes  that  the  first  series  of 
guinea-pigs  succumbed  because  of  the  impossibility  of  fighting  against 
the  typhoid  bacillus  by  means  of  the  free  cytase,  this  being  neutralised 
by  the  anticytase  serum.  The  fact  pointed  out  by  Wassermann  is 
perfectly  accurately  stated  and  has  been  confirmed  by  Besredka2,  in 
an  investigation  carried  out  in  my  laboratory.  Nevertheless,  it  is 
impossible  to  accept  Wassermann's  view  as  to  the  part  played  by  [202] 
anticytase  in  his  experiment.  As  clearly  demonstrated  by  Besredka, 
the  anticytase  serum  acts  not  merely  by  neutralising  the  bactericidal 
ferment,  but  also  by  its  other  properties,  especially  by  one  which 
prevents  the  stimulation  of  the  phagocytes. 

In  the  struggle  of  the  guinea-pig's  organism  against  a  strong 
dose  of  typhoid  cocco-bacilli  (in  Wassermann's  experiments  40  times 
the  lethal  dose),  the  free  cytase  plays  a  part  so  infinitely  small  that 
even  the  injection  into  a  guinea-pig  of  a  large  quantity  of  serum  (3  c.c.) 
from  a  normal  guinea-pig  (containing  much  cytase)  does  not  prevent 
the  death  of  the  animal.  It  is  only  the  blood  serum  of  other  species 
(rabbit  or  ox)  that  is  capable  of  protecting  a  guinea-pig  against  such 
a  large  quantity  of  typhoid  bacilli. 

Wassermann  was  in  error  in  supposing  that  his  experiment  was  a 
case  of  natural  immunity.  It  comes  entirely  within  the  range  of  the 
phenomena  of  acquired  immunity.  In  fact,  the  natural  immunity 
of  the  guinea-pig  is  only  exhibited  against  a  dose  40  times  less  than 

1  Deutsche  med.  Wchmchr.,  Leipzig,  1901,  S.  4. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  209. 


192  Chapter  VII 

that  employed  by  Wassermann.  Consequently  the  control  guinea- 
pigs  which  received  such  a  huge  quantity  of  the  typhoid  cocco-bacilli, 
going  beyond  40  times  the  limit  of  their  natural  immunity,  require 
to  be  preserved  from  death  by  the  injection  of  a  large  quantity  of 
blood  serum  heated  to  60°  C.  from  the  normal  rabbit.  This  serum, 
deprived  of  its  cytase,  retains  its  other  properties,  by  which  the 
organism  of  the  guinea-pig  profits,  especially  exercising  a  stimulating 
action  on  the  phagocytes  of  the  guinea-pig.  The  immunity  of  Wasser- 
manri's  control  animals  was,  then,  really  an  acquired  immunity,  the 
result  of  the  introduction  into  their  organism  of  the  stimulating 
serum  of  the  rabbit.  For  this  reason  an  analysis  of  the  work  of  this 
observer  must  be  postponed  until  we  treat  of  the  phenomena  of 
acquired  immunity  under  the  influence  of  normal  serums. 

We  must,  then,  persist  in  the  opinion  that  the  plasmas  of  the 
normal  animal,  containing  no  cytases,  cannot  play  a  bactericidal  part 
in  natural  immunity,  a  part  which  devolves  upon  the  cytase  contained 
within  the  phagocytes. 

This  result  accords  well,  also,  with  the  whole  of  the  facts  bearing 
on  the  destruction  of  micro-organisms  in  the  animal  body.  The 
transformation  into  granules  of  the  attenuated  cholera  vibrios  that 
is  sometimes  observed  in  the  peritoneal  cavity  during  the  period  of 
phagolysis,  and  the  absence  of  this  transformation  under  conditions 
where  the  peritoneal  leucocytes  are  protected  against  this  injury,  is 
[203]  clearly  explained.  In  the  first  case,  Pfeiffer's  phenomenon  is  set  up 
by  the  bactericidal  substance  which  has  escaped  from  the  leucocytes 
that  have  been  altered  by  the  foreign  substances  injected  into  the 
peritoneal  cavity ;  in  the  second  case,  this  phenomenon  is  not  pro- 
duced because  the  leucocytes  remain  intact.  The  absence  of  this 
granular  transformation  in  the  anterior  chamber  of  the  eye  and  in 
the  subcutaneous  tissue  is  also  readily  explained  by  the  fact  that  the 
bactericidal  substance,  not  being  present  in  the  blood  plasma,  cannot 
pass  into  the  exudations  of  the  eye  and  subcutaneous  tissue1. 

1  Since  Nuttall's  first  paper  appeared  a  certain  bactericidal  action  of  the  aqueous 
humour  has  been  observed.  This  fact  should  be  taken  into  consideration  in  the 
study  of  the  question  of  the  phagocytic  origin  of  the  bactericidal  substance  of  the 
body  fluids.  If  this  substance  really  comes  from  the  phagocytes,  it  should  not  be  found 
in  the  transparent  aqueous  humour  that  contains  no,  or  almost  no,  leucocytes.  Now 
this  fluid  sometimes  destroys  a  certain  number  of  micro-organisms.  This  apparent 
contradiction  is  explained  by  the  fact  that  the  bactericidal  action  may  be  exercised 
by  all  kinds  of  fluids,  such  as  physiological  salt  solution,  nutritive  broths,  etc.  The 
bactericidal  property  of  the  aqueous  humour  comes  into  this  category.  Its  action  is, 


Mechanism  of  immunity  against  micro-organisms    193 

The  bactericidal  substance,  then,  is  essentially  some  substance 
•which  remains  inside  the  uninjured  phagocytes  in  the  living  animal 
but  which  escapes  from  these  cells  when  they  are  injured,  either  in 
the  body  of  the  animal  or  outside  in  the  blood  withdrawn  from  the 
organism.  Buclmer  has  given  to  this  substance  the  name  of  alexine 
and  we  must  now  determine  whether  this  substance  is  the  same 
cytase  which  digests  the  formed  elements  on  their  resorption. 

Since  his  first  researches  on  the  power  of  one  normal  blood  serum  [204] 
to  dissolve  the  red  corpuscles  of  another  species,  Buchner1  has  main- 
tained the  identity  of  the  haemolytic  substance  with  the  bactericidal 
substance  of  the  same  serum.  In  both  cases  we  have  to  do,  according 
to  him,  with  one  and  the  same  substance  of  an  albuminoid  nature, 
with  the  same  "alexine."  In  his  later  work,  Buchner  attempted 
to  confirm  and  develop  this  thesis.  Bordet2  has,  on  several  occa- 
sions, brought  forward  arguments  in  favour  of  the  same  view ;  but 
against  this  Ehrlich  and  Morgenroth3  have  declared  themselves.  Ac- 
cording to  these  observers  a  single  serum  may  contain  several 
alexines  or  "complements."  The  same  serum  may  even  contain 
two  complements,  one  of  which  is  destroyed  by  heating  to  55°  C., 
whilst  the  other,  much  more  stable  as  to  the  action  of  heat,  resists 
this  temperature.  In  one  of  their  most  recent  memoirs,  Ehrlich 

as  a  rule,  much  more  feeble  than  the  action  of  serums  and  exudations  and  is  not 
modified  by  heating  to  55° — 56°  C.  In  certain  aqueous  humours,  a  little  cytase, 
or  true  bactericidal  substance,  may  come  into  play,  for  we  find  aqueous  humours 
which  coagulate  and  which,  when  centrifugalised,  show  a  small  deposit  of  leucocytes. 
These  results  have  been  obtained  by  Mme.  Metchnikoff. 

It  must  not  be  forgotten  also  that,  even  in  the  bactericidal  action  of  blood 
serums,  a  certain  factor  is  the  change  of  medium  which  the  micro-organisms  ex- 
perience with  the  plasmolytic  phenomena  which  follow.  But  it  is  not  possible  to 
ascribe  to  this  factor  the  whole  of  the  bactericidal  property  of  serums  and  exudations, 
as  is  done  by  Baumgarten  (Arb.  a.  d.  pathol.-anat.  Inst.  zu  Tubingen,  1899,  Bd.  in, 
S.  1,  and  Berl.  klin.  IVchnschr.,  1900,  SS.  136, 162, 192),  and  his  pupils  Jetter  and  Walz 
supported  by  A.  Fischer  (Ztschr.f.  Hyg.,  Leipzig,  1900,  Bd.  xxxv,  S.  1).  The  idea  of 
reducing  the  destruction  of  bacteria  in  serums  and  exudations  to  the  effect  of  osmotic 
pressure  has  been  recently  elaborately  analysed  by  v.  Lingelsheim  (Ztschr.  f.  Hyg., 
Leipzig,  1901,  Bd.  xxxvn,  S.  131).  With  great  justness  he  comes  to  the  conclusion  that 
"  the  existence  in  extravascular  blood  or  in  serum,  of  bactericidal  substances  acting 
as  soluble  ferments  can  now  no  longer  be  denied  "  (p.  167).  In  studying  this  question 
we  must  not  lose  sight  of  the  fact  that  these  bactericidal  substances  (alexines, 
complements,  or  cytases)  give  rise  to  the  production  in  the  animal  organism  of 
antagonistic  substances  as  described  by  us  in  the  5th  Chapter. 

1  Verhandl.  d.  Congresses/,  inn.  Med.,  Wiesbaden,  1892,  S.  273. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1900,  t.  xiv,  p.  257 ;  1901,  t  xv,  p.  312. 

3  Berl  klin.  Wchnschr.,  1900,  SS.  453,  677. 

B.  13 


194  Chapter  VII 

and  Morgenroth  lay  special  stress  on  the  importance  of  an  experi- 
ment which  has  enabled  them,  by  means  of  filtration,  to  separate 
two  complements  from  the  normal  serum  of  the  goat,  one  of  them 
attacking  the  red  corpuscles  of  the  guinea-pig,  the  other  those  of  the 
rabbit. 

Max  Neisser1  has  adopted  this  view  of  the  plurality  of  alexines. 
According  to  Ehrlich  and  Morgenroth,  the  same  serum  may  possess 
several  complements  which  attack  the  red  blood  corpuscles  of  various 
species  and  other  complements  which  attack  micro-organisms.  In 
favour  of  this  thesis  Neisser  gives  a  summary  of  his  experiments  on 
the  absorption  of  complements  which,  in  his  opinion,  prove  the 
plurality  of  alexines.  By  centrifugalising  rabbit's  blood  serum  to 
which  he  had  previously  added  a  certain  number  of  anthrax  bacilli, 
he  obtained  a  fluid  which  no  longer  destroyed  this  bacillus  but 
which  still  dissolved  the  red  corpuscles  of  goat  and  sheep.  There 
are  then,  according  to  Neisser,  in  the  normal  serum  of  the  rabbit,  at 
least  two  different  complements  ;  one  for  the  bacilli  and  one  for  the 
red  corpuscles. 

With  the  object  of  explaining  the  discrepancy  between  these 
results  and  those  of  his  previous  experiments,  Bordet2  undertook 
[205]  a  new  series  of  researches  on  the  absorption  of  cytases.  He  first 
made  it  clear  that  the  normal  red  corpuscles,  when  plunged  into  a 
normal  haemolytic  serum,  are  incapable  of  fixing  all  the  cytase. 
When  such  a  serum  is  centrifugalised,  after  a  prolonged  contact 
with  red  corpuscles  of  a  different  species,  the  fluid  no  longer  dissolves 
normal  red  corpuscles.  But  if  these  latter  be  sensibilised  by  means 
of  a  specific  fixative,  the  red  corpuscles  are  dissolved  in  large 
numbers.  It  must  be  admitted  that  in  this  experiment  we  have 
to  do  with  a  single  cytase  because,  before  ceutrifugalisation,  as  after 
it,  the  red  corpuscles  of  the  same  species  are  added.  In  the  first 
case,  however,  these  corpuscles  were  normal,  whilst  in  the  second 
they  were  sensibilised  by  the  fixative. 

When,  after  the  first  part  of  this  experiment,  that  is  to  say,  after 
the  fixation  of  a  certain  quantity  of  cytase  by  the  red  corpuscles,  we 
centrifugalise  the  mixture  and  add,  not  the  sensibilised  red  corpuscles 
of  the  same  species  but  the  normal  red  corpuscles  of  a  different 
species,  we  find  that  the  latter  still  dissolve  and  fix  a  certain  quantity 
of  cytase.  As  the  first  experiment  (with  sensibilised  red  corpuscles) 

1  Deutsche  med.  Wchnschr.,  Leipzig,  1900,  S.  790. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  303. 


Mechanism  of  immunity  against  micro-organisms    195 

has  shown  that  the  whole  of  the  cytase  has  not  been  absorbed  by 
the  red  corpuscles,  we  readily  understand  that  the  portion  re- 
maining in  the  fluid  will  act  on  the  normal  red  corpuscles  of  another 
species. 

But  when  we  fix  the  cytase  to  the  sensibilised  red  corpuscles  the 
absorption  becomes  complete  and  the  addition  of  other  species  of 
red  corpuscles  no  longer  produces  any  solution.  It  is  easy,  therefore, 
by  means  of  sensibilised  red  corpuscles,  to  take  out  the  whole  of  the 
cytase  from  a  serum.  When  to  such  a  serum,  thus  deprived  of  the 
whole  of  its  haemolytic  cytase,  we  add  bacteria,  these  latter  show  no 
sign  of  disintegration ;  whilst  previously,  that  is  before  the  absorption 
of  the  cytase  by  the  sensibilised  red  corpuscles,  the  same  serum  was 
highly  bactericidal.  Let  us  take  a  concrete  example  so  that  the  reader 
may  form  some  definite  idea  of  the  phenomena  observed.  Take  a 
normal  rat's  serum  which,  in  a  very  short  time,  transforms  cholera 
vibrios  into  granules  or  deforms  and  dissolves  anthrax  bacilli.  The 
same  serum  dissolves  the  red  corpuscles  of  a  different  species.  We 
will  first  leave  this  serum  in  contact  with  these  red  corpuscles  sensi- 
bilised by  the  specific  fixative.  After  the  solution  of  a  quantity  of 
these  red  corpuscles,  let  us  add  to  the  serum  a  few  cholera  vibrios 
or  anthrax  bacilli.  The  vibrios,  in  this  serum,  are  no  longer  trans- 
formed into  granules  and  the  anthrax  bacilli  undergo  no  change  at 
all;  they  stain  in  the  normal  fashion  by  basic  aniline  dyes,  they [206] 
present  neither  deformations  nor  solution  of  their  contents.  In 
other  words,  no  bactericidal  action  takes  place  in  a  serum  that  is 
deprived  of  its  cytase  by  sensibilised  red  corpuscles. 

Is  it  necessary  to  conclude  from  this  and  other  analogous  experi- 
ments that  the  cytase,  fixed  by  the  sensibilised  formed  elements  (red 
blood  corpuscles  or  micro-organisms),  is  always  one  and  the  same 
cytase  ?  May  it  not  be  that  these  elements,  impregnated  with  specific 
fixatives,  become  so  greedy  for  cytases  that  it  is  easy  for  them  to 
absorb  not  only  one  variety  but  several  species  of  cytases  ? 

The  facts  we  have  summarised  in  Chapter  IV  concerning  the 
cytases,  indicate  that  very  probably  there  exist  two  kinds  of  cytases, 
connected  with  the  two  great  groups  of  phagocytes.  Extracts  of 
the  mesenteric  glands,  of  the  omentum  and  of  the  exudations,  which 
are  composed  for  the  most  part  of  microphages,  do  not  dissolve  the 
red  corpuscles,  but  are,  on  the  other  hand,  specially  bactericidal. 
Sarassewitch  has  carried  out  numerous  experiments  on  this  point 
in  my  laboratory  and  has  brought  forward  a  large  number  of  data 

13—2 


196  Chapter  VII 

in  favour  of  this  theory  of  two  phagocytic  cytases.  He  found  that, 
even  when  specific  fixative  is  added  to  the  extract  of  microphagic 
exudations  (of  rabbit),  the  seusibilised  red  corpuscles  are  not  dis- 
solved. It  must  then  be  accepted  that  microcytase,  so  active  against 
bacteria,  is  entirely  powerless  against  animal  cells. 

As  the  microphages  seize,  though  rarely,  and  digest  red  blood 
corpuscles,  spermatozoa  and  other  cells  of  animal  origin,  it  must  be 
admitted  that  they  also  contain  a  small  quantity  of  macrocytase,  or 
that  the  microcytase,  given  time,  is  capable  of  dissolving  these  ele- 
ments. On  the  other  hand,  the  macrophages,  in  spite  of  their  marked 
predilection  for  animal  cells,  also  ingest  and  digest  certain  bacteria. 
This  is  due  perhaps  to  the  presence  of  a  little  microcytase  or  to  the 
power  that  the  macrocytase  has  of  attacking  micro-organisms.  These 
questions  are  too  subtle  to  be  definitely  resolved  at  present. 

The  duality  of  the  cytases  does  not  clash  with  the  experiments  of 
Bordet  summarised  above.  We  have  only  to  admit  that  the  formed 
[207]  elements,  once  they  are  impregnated  with  specific  fixatives,  become 
capable  of  absorbing  not  only  the  cytase  which  digests  them,  but  also 
another  which,  without  dissolving  them,  is  simply  fixed  to  them.  Here 
we  should  have  a  phenomenon  analogous  to  the  fixation  by  fibrin 
of  diastases,  other  than  trypsin  and  pepsin,  or  to  the  fixation  by  silk 
threads  of  all  kinds  of  soluble  ferments. 

It  may  be  accepted,  then,  that  the  phagocytes  elaborate  two 
cytases :  macrocytase,  active  for  animal  cells,  and  microcytase,  which 
digests  bacteria.  This  result  up  to  a  certain  point  has  been  an- 
ticipated by  Schattenfroh's1  experiments  and  foreseen  by  Max  Neisser 
(Z.c.). 

It  has  already  been  noted  that  the  reaction  inside  the  phagocytes 
is  usually  feebly  or  very  feebly  acid,  and  only  rarely  distinctly  al- 
kaline. On  the  other  hand,  it  is  well  known  that  cytases,  in  serums, 
act  in  an  alkaline  medium.  It  is  certain  therefore  that  these  soluble 
ferments  can  carry  on  the  process  of  digestion  under  varied  con- 
ditions. Hegeler2,  working  in  Buchner's  laboratory,  has  studied 
the  influence  of  the  alkalinity  and  acidity  of  the  medium  on  the 
bactericidal  action  of  serum.  He  comes  to  the  conclusion  that  the 
destruction  of  micro-organisms  can  take  place  in  a  serum  to  which 
has  been  added  small  quantities  of  alkali  (carbonate  of  soda)  and 
also  in  a  weakly  acid  serum  (from  the  addition  of  small  quantities 

1  Arch./.  Hyg.,  Miinchen  u.  Leipzig,  1899,  Bd.  xxxv,  S.  199. 

2  Arch.f.  Hyg.,  Miinchen  u.  Leipzig,  1901,  Bd.  XL,  S.  375. 


Mechanism  of  immunity  against  micro-organisms     197 

of  sulphuric  acid).     Once  the  serum  becomes  distinctly  acid  the 
bactericidal  power  disappears  at  once. 

Our  knowledge  of  the  cytases,  as  a  whole,  leads  us  to  approximate 
these  diastases  to  the  group  of  trypsins,  papain,  amoebodiastase  and 
actinodiastase.  The  cytases  are  elaborated  by  the  phagocytes,  but 
are  not  secreted  into  the  plasmas  and  they  remain  inside  the  cells  so 
long  as  these  cells  remain  uninjured. 

In  this  respect  the  cytases  must  be  placed  in  the  group  of  the 
"  Endo-enzymes,"  according  to  the  nomenclature  of  Hahn  and  Geret1. 
These  observers  have  carefully  studied  the  proteolytic  diastase  of  the 
yeast  of  beer  which  likewise  acts  inside  the  cells  without  ever  being 
excreted.  This  diastase,  to  which  they  give  the  name  of  "yeast  endo- 
trypsin"  (Hefeendotrypsin),  presents  in  general  an  undeniable  rela- 
tionship with  the  phagocytic  cytases,  from  which  it  is  distinguished  [208] 
however  by  a  greater  sensitiveness  to  alkalis.  Kutscher2  in  his 
researches  on  autodigestion  in  yeast  has  established  analogous  facts. 

The  cytases  and  endotrypsin  are  consequently  endo-enzymes,  as 
are  also  amoebodiastase,  actinodiastase,  plasmase  (fibrin  ferment) 
and  the  zymase  of  E.  Buchner.  All  remain  confined  within  the  cells 
which  have  manufactured  them  and  are  not  secreted  or  excreted,  as 
are  the  sucrase  and  invertin  produced  by  yeasts  or  Mucediuae. 

Our  present  knowledge  on  the  cytases  is  as  yet  far  from  perfect, 
which  is  not  astonishing,  seeing  how  recently  the  question  has  been 
brought  forward.  The  cytases  found  in  the  serum  of  the  same  animal 
are  the  same,  for  we  have  seen  that  the  macrocytase  which  dissolves 
red  blood  corpuscles  is  the  same  which  digests  spermatozoa ;  whilst 
the  same  microcytase  digests  bacilli,  spirilla,  and  cocci.  But  in  the 
serums  of  different  species,  the  cytases  differ.  Thus  the  cytases  of 
the  dog  are  not  the  same  as  are  those  found  in  the  serums  of  the 
rabbit  or  horse.  Whilst  the  majority  of  the  cytases  are  very  sensitive 
to  heat  and  are  destroyed  at  a  temperature  of  55° — 56°  C.,  some, 
e.g.  the  microcytase  of  rat's  serum,  resist  this  temperature  and  are 
only  destroyed  at  65°  C.,  presenting,  consequently,  an  example  of 
cytase  stable  to  heat  similar  to  that  discovered  by  Ehrlich  and 
Morgenroth. 

It  is  as  yet  very  difficult  to  establish  whether,  besides  the  cytases, 
there  exist  other  endo-enzymes  within  phagocytes,  that  is  to  say, 
soluble  ferments  which  do  not  pass  into  the  serums  on  the  destruction 

1  Ztschr.  f.  £iol.,  Miinchen  u.  Berlin,  1900,  Bd.  XL,  S.  117. 

2  Sitzungsb.  d.  naturforsch.  Gesellsch.  zu  Marburg,  1900. 


198  Chapter  VII 

of  the  phagocytes,  but  continue  within  these  cells.  Our  present 
methods  of  investigation  do  not  enable  us  to  come  to  any  conclusion 
on  this  point.  We  know  only  that  the  digestion  of  the  formed 
elements  is  more  complete  inside  the  phagocytes  than  in  the  serums. 
Thus,  as  we  have  seen  in  Chapter  IV,  the  best  spermotoxic  and 
haeinolytic  serums  never  digest  either  spermatozoa  or  the  nuclei  of 
the  red  corpuscles  of  birds.  And  yet  these  elements  are  completely 
dissolved  in  the  phagocytic  contents.  Does  this  difference  depend 
on  the  fact  that,  in  the  serums,  we  get  only  a  very  small  part  of  the 
[209]  macrocytase,  or  upon  the  injurious  influence  of  the  alkalinity  of  the 
serums  on  the  macrocytase  which  acts  better  in  weakly  acid  media, 
or  on  the  presence  in  the  phagocytes  of  other  endo-enzymes  still  un- 
known ?  These  are  questions  to  which  at  present  no  definite  answer 
can  be  given. 

Just  as  animal  cells,  when  ingested  by  phagocytes  during  resorp- 
tion  (see  Chap.  IV),  immediately  become  permeable  to  stains,  so  in 
natural  immunity  do  micro-organisms  taken  into  phagocytes  acquire 
the  same  property.  Very  often,  under  the  influence  of  the  phagocytic 
action,  the  ingested  micro-organisms  become  stainable  by  eosin  (fig. 
36).  This  eosinophile  transformation  has  been  observed  in  the  cholera 
vibrio,  the  anthrax  bacillus  and  in  Proteus  vulgaris.  It  is  probably 
widely  diffused  among  the  phagocytised  bacteria.  This  fact  demon- 
strates clearly  that  at  least  some  of  the  eosinophile  granules  are 
derived  from  foreign  bodies  ingested  by  the  phagocytes.  Others  of 
these  granules  are  probably  the  result  of  the  transformation  of  soluble 
substances  absorbed  by  the  phagocytes.  In  fact,  during  inflammation, 
many  microphages  which  contain  no  foreign  solid  body,  may  often  be 
seen  charged  with  a  quantity  of  small  pseudo-eosinophile  granules. 

Certain  vibrios  and  bacilli,  when  ingested  by  microphages,  become 
transformed,  almost  immediately,  into  spherical  granules.  The  cholera 
vibrio  undergoes  the  same  transformation  in  the  peritoneal  exudation 
at  the  moment  of  phagolysis,  as  also  in  blood  serum.  The  Bacillus 
coli,  the  typhoid  bacillus,  and  certain  other  cocco-bacilli  do  not  change 
in  the  least,  or  change  very  slightly  in  serum,  but  exhibit  the  trans- 
formation into  granules  when  inside  microphages.  The  macrophages, 
on  the  other  hand,  digest  the  same  bacteria  (vibrios  and  cocco-bacilli) 
without  these  bacteria  presenting  any  signs  of  this  change  of  form. 
The  bacterial  membrane  resists  the  influence  of  the  phagocytic  diges- 
tion longer  than  do  the  contents,  but  is  in  the  long  run  also  completely 
digested.  After  the  ingestion  and  destruction  of  micro-organisms 


Mechanism  of  immunity  against  micro-organisms    199 

by  the  phagocytes,  debris  of  indeterminate  form  may,  for  long,  be 
found  in  the  cells,  but  I  have  never  been  able  to  demonstrate  any 
solid  excreta  from  them.  We  must  suppose,  then,  that  the  undigested 
portions  are  not  thrown  out  from  the  phagocytes. 

When  describing  the  solution  of  red  blood  corpuscles  by  normal 
serums,  we  have  mentioned  Ehrlich  and  Morgenroth's  view  that 
the  cytases  are  incapable  of  fixing  themselves  to  these  cells  with-  [210] 
out  the  help  of  fixatives.  They  cite  in  support  of  their  opinion 
several  examples  of  fixatives  (intermediary  substances  or  "  Zwischen- 
kb'rper")  discovered  by  them  in  the  serums  of  various  species  of 
mammals.  Is  this  so  with  microcytase  in  respect  to  micro-organisms  ? 
If  this  soluble  ferment  is  incapable  alone  of  fixing  itself  upon  the 
bodies  of  these  parasites,  the  help  of  fixatives  would  be  indispensable 
to  it.  The  bactericidal  property  of  the  microcytase,  then,  would 
depend  on  the  existence  of  another  body  (fixative)  which,  perhaps, 
might  not  owe  its  origin  to  phagocytes.  The  problem,  then,  has  a 
wide  general  range. 

In  one  of  his  memoirs,  Bordet1  had  already  raised  the  question  of 
the  existence  of  this  sensibilising  (or  fixative)  property  in  normal 
serums.  By  mixing  two  normal  serums  coming  from  different  species, 
he  was  sometimes  able  to  demonstrate  the  existence  of  such  fixatives. 
Thus  the  cholera  vibrios,  which  do  not  undergo  granular  transforma- 
tion in  either  the  normal  serum  of  the  horse  (which  is  capable  only 
of  arresting  their  movements  and  agglutinating  them  into  a  mass) 
or  in  that  of  the  normal  guinea-pig,  readily  become  transformed  into 
granules  when  placed  in  contact  with  a  mixture  of  the  two  serums. 
Bordet,  however,  refrains  from  any  hasty  generalisation  on  this 
observation  and  proposes  to  make  fresh  researches  on  this  subject. 
Independently,  Moxter2  has  attempted  to  demonstrate  the  presence 
of  fixative  in  the  normal  serum  of  the  guinea-pig.  When  deprived 
of  cytases  by  heat,  this  serum  is  incapable  of  transforming  the  cholera 
vibrios  into  granules ;  but  when  fluid  from  the  peritoneal  exudation 
of  the  same  guinea-pig  is  added,  the  transformation  takes  place 
very  rapidly.  Nevertheless,  as  this  exudation  was  already,  by  itself, 
capable  of  producing  Pfeifler's  phenomenon,  Moxter's  conclusions  on 
the  presence  of  the  fixative  in  the  normal  guinea-pig's  serum  cannot 
be  accepted  without  a  fuller  analysis  of  the  facts,  and  this  demands 
fresh  researches. 

1  Ann.  de  VInst.  Pasteur,  Paris,  1899,  t.  xm,  p.  295. 

2  Centralbl.f.  Bakteriol  u.  Parasitenk.,  Jena,  1899,  Ite  Abt,  Bd.  xxvi,  S.  344. 


200  Chapter  VII 

A  recent  investigation,  carried  out  by  Bordet1  in  collaboration 
with  Gengou,  devoted  to  the  study  of  the  absorption  of  cytases 
by  micro-organisms  that  have  been  sensibilised  by  means  of  fixa- 
tives, also  gives  us  information  on  the  question  which  now  occupies 
[211]  us.  It  was  easy  to  demonstrate  the  presence  of  fixative  in  the 
serums  in  the  case  of  the  cholera  vibrio  and  its  allies,  by  reason 
of  their  transformation  into  granules,  appreciable  on  microscopical 
examination.  When  a  serum,  which  of  itself  is  incapable  of  set- 
ting up  this  transformation,  produces  it  directly  we  add  another 
serum  heated  to  55°  C.,  we  must  conclude  that  the  latter  fluid 
contains  the  cholera  fixative,  whilst  the  former  contains  only  cytases. 
But,  as  the  majority  of  bacteria  do  not  undergo  any  analogous  trans- 
formation in  serums,  we  are,  in  these  cases,  without  any  criterion  as 
to  the  presence  of  fixative.  Bordet  and  Geugou  have  eliminated  this 
inconvenience  in  determining  the  fixation  of  alexine  by  bacteria 
which  undergo  neither  granular  transformation  nor  any  other  visible 
change.  A  normal  unheated  serum,  which  always  contains  a  sufficient 
quantity  of  cytases,  is  mixed  with  any  micro-organism,  e.g.  with  the 
anthrax  bacillus  or  the  cocco-bacillus  of  plague.  The  serum,  decanted 
after  a  prolonged  contact  with  these  bacteria,  remains  quite  as  capable 
of  dissolving  the  red  corpuscles  of  a  determined  foreign  species  as  it 
was  originally.  This  proves  that  cytases  remain  in  the  serum  and 
that  they  have  not  been  absorbed  by  the  bacteria.  Repeat  the  same 
experiment  with  this  difference,  that  instead  of  normal  anthrax  bacilli 
or  plague  cocco-bacilli  we  introduce  into  the  unheated  normal  serum 
these  bacteria  after  they  have  been  sensibilised  by  the  corresponding 
fixatives  (that  is  to  say,  previously  submitted  to  the  influence  of  specific 
serums  heated  to  55°  C.).  After  contact  for  a  certain  length  of  time 
with  these  bacteria  the  serum  is  no  longer  capable  of  dissolving  the 
red  corpuscles  of  a  determined  foreign  species,  thus  demonstrating 
that  the  cytases  have,  thanks  to  the  help  of  the  fixatives,  been  linked 
to  the  bacteria.  We  see,  therefore,  that  it  is  easy  to  determine 
whether  a  serum,  whose  properties  are  unknown,  contains  fixatives 
or  not.  It  is  heated  to  55°  C.  and  mixed  with  normal  unheated  serum 
to  which  bacteria  are  added.  If,  after  contact  with  these  latter 
the  normal  serum  has  lost  the  power  of  dissolving  the  red  corpuscles 
(which  .it  was  capable  of  dissolving  previously),  it  is  because  its 
cytases,  thanks  to  the  fixative  which  must  be  present  in  the  heated 

1  Ann.  de  I'Inst.  Pasteur,  Paris,  1901,  t.  xv,  p.  289. 


Mechanism  of  immunity  against  micro-organisms    201 

serum,  have  been  absorbed  by  the  bacteria.    In  the  other  case,  we 
conclude  the  non-existence  of  the  fixative. 

In  their  researches,  Bordet  and  Gengou  often  employed  normal 
unheated  serums  to  which  they  added  several  species  of  bacteria. 
They  demonstrated  that  in  these  mixtures  the  cytases  remained  [212] 
intact  or  nearly  so.  These  soluble  ferments  were  scarcely,  if  at 
all,  absorbed  by  the  bacteria,  which  proves  that  in  the  normal 
serums  there  are  no  fixatives  in  any  appreciable  quantity.  Of  all 
their  experiments  the  one  that  interests  us  most  was  carried  out 
with  Proteus  vulgar  is.  This  organism  placed  in  prolonged  contact 
with  normal  guinea-pig's  serum  showed  itself  incapable  of  absorbing 
or  fixing  anything  beyond  the  most  minute  quantities  of  the  cytases. 
There  is  consequently  no  fixative  for  Proteus  in  normal  guinea-pig's 
serum,  or,  if  any  exists,  it  is  only  in  negligible  quantity.  And  yet 
this  same  Proteus  vulgaris,  when  injected  into  guinea-pigs,  was  in 
a  short  time  ingested  and  destroyed  by  the  phagocytes  which  assure 
to  the  animal  a  natural  immunity  of  the  most  stable  character.  The 
facility  with  which  the  leucocytes  of  the  guinea-pig  devour  the 
Proteus  follows,  among  others,  from  an  experiment  by  Bordet1 
carried  out  with  quite  another  object.  A  guinea-pig,  very  ill  as  the 
result  of  the  injection  into  its  peritoneal  cavity  of  a  very  virulent 
streptococcus,  contained  in  the  peritoneal  exudation  a  quantity  of 
empty  microphages  incapable  of  ingesting  these  streptococci.  At 
this  critical  moment  there  was  injected  into  the  same  position  a 
mass  of  Proteus  vulgar  is.  "  At  the  end  of  a  very  short  time,  it  is 
seen  that  the  leucocytes  which  energetically  refuse  to  ingest  strepto- 
cocci greedily  seize  upon  the  new  organism  offered  to  them  ;  and  at 
the  end  of  half-an-hour  the  whole  of  these  organisms  are  found  in- 
side phagocytes." 

Here,  then,  we  have  an  actual  proof  of  the  fact  that  the  phago- 
cytes, in  order  to  rid  the  animal  organism  of  a  microbe  and  assure 
to  it  a  natural  immunity,  have  no  need  of  any  previous  help  from  an 
extraphagocytic  fixative.  The  phagocytes  act,  so  to  speak,  motu, 
2)roprio,  and  themselves  bring  about  the  resorption  of  the  intruders. 
The  question  of  fixatives  in  normal  serums,  then,  loses  its  importance 
for  us  and  their  origin  no  longer  presents  any  essential  interest  for 
the  problem  with  which  we  are  at  present  occupied. 

Can  we  conclude,  from  the  data  just  summarised,  that  the  cytases, 

1  Ann.  de  Vlnst.  Pasteur,  Paris,  1896,  t.  x,  p.  107. 


202  Chapter  VII 

which  in  several  respects  approximate  to  the  trypsins,  have  this 
further  feature  in  common  with  them  that  they  can  act  without  the 
help  of  any  fixative  ?  It  is  known,  as  mentioned  in  Chapter  III,  that 
trypsin  can  digest  alone,  or  in  collaboration  with  enterokynase,  that 
ferment  of  the  intestinal  juice  which  acts  as  such  a  powerful  ad- 
[2i3]juvant  to  the  pancreatic  ferments.  Is  this  also  the  case  with  the 
cytases?  The  fact  that  when  Proteus  vulgaris  is  placed  in  contact 
with  normal  unheated  guinea-pig's  serum,  it  is  incapable  of  absorbing 
cytases,  although  it  is  so  readily  digested  by  phagocytes,  indicates 
rather  that,  for  the  fixation  of  cytases,  the  help  of  the  fixative  is 
indispensable.  But,  as  this  fixative  is  absent  from  the  serum,  and 
since,  nevertheless,  it  must  exist  for  the  needs  of  digestion,  it  must 
clearly  be  concluded  that  it  is  found  inside  the  phagocytes.  Its 
quantity  is  perhaps  so  small  that  when  it  has  passed  into  the  serum 
its  action  is  entirely  lost  or  nearly  so.  Fresh  researches  are  necessary 
to  elucidate  this  delicate  point. 

But  perhaps  the  phagocytes  which,  as  we  have  just  seen,  can 
engage  in  a  struggle  with  and  ingest  the  micro-organisms  without  the 
latter  being  previously  modified  by  the  fixative,  may  be  incapable 
of  fulfilling  their  functions  without  the  help  of  some  other  substance 
circulating  in  the  blood  plasma?  Amongst  these  substances  is  one 
which  manifestly  acts  upon  the  micro-organisms  by  rendering  them 
motionless  and  agglomerating  them  into  masses.  This  agglutinative 
property  is  met  with  in  the  normal  fluids  of  many  species  of 
animals  and  is  exercised  upon  many  bacteria.  It  may  be  demon- 
strated not  only  in  the  blood  serum,  but  also  in  the  fluids  of 
transudations  and  exudations  and  in  certain  secretions  such  as 
milk,  tears,  and  urine.  Little  is  known  as  yet  of  the  mechanism  of 
this  agglutinative  action,  and  we  can  the  more  readily  refrain  from 
entering  into  details  concerning  it  as  it  is  of  no  great  importance 
from  the  point  of  view  of  natural  immunity. 

In  the  preceding  chapter  we  have  already  spoken  of  the  ingestion 
of  cholera  vibrios  in  the  peritoneal  cavity  of  guinea-pigs.  In  those 
cases  in  which  the  animals  exhibit  an  effective  resistance,  the 
phagocytes  devour  the  vibrios  whilst  they  still  exhibit  very  active 
movements.  Even  when  a  large  majority  are  already  seized  by  the 
leucocytes  and  only  a  few  isolated  free  vibrios  remain,  these  latter 
still  continue  to  exhibit  normal  movements.  These  facts,  repeatedly 
observed,  clearly  demonstrate  that  phagocytosis  may  take  place 
without  any  previous  agglutinative  action  ;  this  does  not,  however, 


Mechanism  of  immunity  against  micro-organisms    203 

prevent  the  micro-organisms,  when  united  into  motionless  masses, 
from  being  ingested  by  the  leucocytes  with  greater  ease. 

In  the  case  of  the  typhoid  bacillus,  one  of  the  most  active  of  bacteria, 
the  same  facts  may  be  observed  as  in  the  case  of  the  cholera  vibrio.  [214] 
In  animals  that  remain  unaffected  we  often  see  the  last  free  bacilli 
moving  about  actively  between  the  leucocytes  filled  with  microbes. 
In  many  other  examples  of  natural  immunity  we  constantly  meet 
with  phagocytes  containing  but  a  single  or  a  small  number  of  micro- 
organisms (streptococci,  yeasts,  etc.). 

The  presence  of  motile  micro-organisms  inside  phagocytes  proves 
also  that  it  is  possible  for  these  cells  to  do  without  the  help  of  agglu- 
tinative substance  in  carrying  on  their  protective  work.  The  most 
carefully  studied  case  of  the  relations  between  natural  immunity  and 
agglutination  is  that  met  with  in  the  anthrax  bacillus.  We  owe  it  to 
Gengou1,  who  at  the  Liege  Bacteriological  Institute  carried  out  a  very 
detailed  investigation  on  this  question.  He  showed  that  the  bacillus 
of  Pasteur's  first  anthrax  vaccine  is  agglutinated  by  the  blood  serum  of 
a  great  number  of  animals.  But  he  also  showed  that  the  serums  which 
have  the  greatest  agglutinative  action  on  this  bacillus  do  not  come  from 
the  most  refractory  species.  Human  serum  agglutinates  most  strongly 
the  bacillus  of  the  first  vaccine  (in  the  proportion  of  one  part  of 
serum  to  500  parts  of  culture)  but  man  is  far  from  being  exempt 
from  anthrax.  Pigeon's  serum,  on  the  other  hand,  is  completely 
without  any  agglutinative  power,  although  this  species  resists  not  only 
the  first  vaccine  but  very  often  even  virulent  anthrax.  The  serum 
of  the  ox,  a  species  susceptible  to  anthrax,  is  more  agglutinative 
(1  :  120)  than  that  of  the  refractory  dog  (1  :  100).  There  are,  how- 
ever, exceptional  cases  in  which  the  agglutinative  property  cor- 
responds to  the  degree  of  susceptibility.  Thus  the  serum  of  the 
mouse  has  not  the  slightest  agglutinative  action  on  the  bacillus  of 
the  first  vaccine.  But  alongside  this  example  is  that  of  the  rat,  a 
species  of  moderate  susceptibility  to  anthrax,  whose  serum  possesses 
the  least  agglutinating  power  of  all,  acting  only  in  the  proportion 
of  1 : 10.  All  these  facts  fully  justify  the  conclusion  formulated  by 
Gengou  that  "  we  cannot  establish  any  relation  between  the  aggluti- 
nating power  and  the  refractory  state  of  the  animals  to  anthrax " 
(p.  319).  This  conclusion  may  be  extended  to  the  phenomena  of  the 

1  Arch,  internat.  de  Pharmacodyn.,  Gand  et  Paris,  1899,  t.  vi,  p.  299;  Ann. 
de  I'Inst.  Pasteur,  Paris,  1899,  t.  xiu,  p.  642. 


204  Chapter  VII 

agglutination  of  micro-organisms  and  to  those  of  natural  immunity 
in  general. 

Amongst  the  properties  of  humours,  there  exists  one  which  might 
play  a  part  in  natural  immunity  against  micro-organisms.  I  mean  the 
[215]  power  possessed  by  the  blood  and  certain  other  fluids  of  the  animal 
body  to  neutralise  the  action  of  microbial  poisons.  Perhaps,  it  may 
be  suggested,  the  phagocytes  are  not  capable  of  commencing  to  do 
their  work  except  after  a  previous  action  of  antitoxins.  After 
the  neutralisation  of  the  principal  means  possessed  by  the  micro- 
organisms to  injure  the  organism,  these  parasites,  having  been  ren- 
dered innocuous,  might  be  readily  destroyed  by  the  phagocytic  cells. 
We  have  already  had  occasion  to  treat  this  fundamental  question. 
Thus,  we  have  insisted  in  the  preceding  chapters  on  the  absence  of 
any  parallelism  between  immunity  against  micro-organisms  and  that 
against  their  toxins,  taking  as  our  examples  anaerobic  bacteria  (tet- 
anus bacillus,  septic  vibrio,  bacillus  of  symptomatic  anthrax)  in  con- 
nection with  which  phagocytosis  takes  place  without  any  help  from 
the  antitoxic  function. 

We  must  now  pass  directly  to  the  examination  of  the  question 
of  antitoxins  in  the  fluids  of  animals  naturally  refractory  to  the 
micro-organisms  and  of  the  ultimate  part  played  by  them  in  this 
immunity. 

Examples  of  the  presence  of  antitoxic  serum  in  normal  animals 
are  very  rare.  It  might  be  supposed  that  animals  endowed  with 
natural  immunity  against  micro-organisms  and  at  the  same  time 
against  their  toxins,  present  an  appreciable  natural  antitoxic  power. 
Let  us  examine  some  of  the  more  typical  examples.  The  fowl  enjoys 
a  very  marked  immunity  against  the  tetanus  bacillus  and  its  toxin  ; 
its  blood  and  its  serum,  however,  as  demonstrated  by  Vaillard1, 
exhibit  no  antitoxic  power ;  this  observation  has  been  confirmed 
by  several  other  workers.  The  rat  is  very  refractory  to  diphtheria ; 
it  resists  subcutaneous  inoculation  of  a  large  quantity  of  diphtheria 
bacilli  and  vigorously  withstands  diphtheria  toxin  when  injected  any- 
where but  into  the  brain.  It  has  been  demonstrated  by  Kuprianow2, 
in  an  investigation  carried  out  under  Loeffler's  direction,  that  the 
blood  serum  and  the  emulsion  of  the  organs  of  the  grey  rat  (Mm 
decumanus)  possess  no  antitoxic  property.  This  fact  has  been  con- 

1  Compt.  rend.  Soc.  de  biol.,  Paris,  1891,  p.  464. 

2  CentralU.f.  Bakteriol.  u.  Parasitenk.,  Jena,  1894,  Bd.  xvi,  S.  415. 


Mechanism  of  immunity  against  micro-organisms    205 

firmed  by  other  observers.  Von  Behring1,  in  a  review  of  the  pheno- 
mena of  immunity  in  general,  sums  up  this  question  as  follows  :  "  we 
find  no  antitoxin  in  the  blood  of  individuals  that  are  naturally 
refractory."  There  are,  however,  certain  exceptions,  perhaps  only  [216] 
apparent,  to  this  rule.  Thus  Wassermann2  has  shown  that  blood 
serum  from  healthy  human  beings  is  sometimes  antitoxic  to  the 
diphtheria  poison.  The  individuals  who  furnished  this  antitoxin 
maintained  that  they  had  never  suffered  from  diphtheria.  We  know, 
however,  that  this  disease  is  sometimes  present  in  so  benign  a  form 
that  it  may  pass  unnoticed.  More  conclusive  appears  the  example 
of  normal  horses  whose  blood  serum,  as  demonstrated  by  Meade 
Bolton3  aiid  Cobbett4,  is  very  often  antitoxic  for  the  diphtheria 
toxin.  This  property,  however,  is  not  found  in  every  horse ;  in 
certain  individuals  it  is  entirely  absent.  This  last  fact  affords  an 
indication  that  the  antitoxic  property  in  the  blood  of  horses  has 
been  acquired  as  the  result  of  some  affection  produced  by  a  bacillus 
allied  to  the  diphtheria  bacillus.  This  view  has  not  yet  been  sufficiently 
examined  and  consequently  cannot  claim  to  be  accepted  as  settled. 
Recently,  Max  Xeisser  and  Wechsberg5  have  discovered  an  antitoxin 
in  human  blood  which  is  capable  of  preventing  the  solution  of  the 
red  corpuscles  by  the  toxin  of  staphylococci.  This  antitoxic  power 
varies  considerably  in  different  individuals  and  is  probably  to  be 
accounted  for  by  the  fact  that  the  staphylococcus  is  one  of  the  most 
widely  diffused  organisms  among  the  bacterial  flora  of  the  human 
body.  The  small  lesions  produced  by  these  micro-organisms  (acne, 
boils,  etc.)  are  so  frequent  in  man  that  they  may  readily  bring  about 
the  production  of  an  antitoxin.  In  this  case,  however,  we  have  again 
an  example  of  acquired  antitoxic  power. 

The  examples  just  summarised  can  in  no  way  affect  the  general 
thesis  that  the  phagocytes,  in  order  to  fulfil  their  microbicidal 
function  in  an  animal  endowed  with  natural  immunity,  have  no  need 
of  any  previous  action  of  the  body  fluids  to  neutralise  the  correspond- 
ing toxins. 

1  Article  "Imnmnitat"  in  the  3rd  edition  of  Eulenburg's  Real-Encydopddie, 
Wien,  1896. 

2  Deutsche  med.  Wchnschr.,  Leipzig,  1894,  S.  120  (of  Vereins-Beilage). 

3  \Journ.  Exper.  Med.,  New  York,  1896,  Vol.  I,  p.  543.] 

4  \Journ.  Path,  and  Bacteriol,  Edin.  and  London,  1896,  Vol.  m,  p.  328 ;  Lancet, 
London,  1899,  Vol.  n,  p.  332;  CentralU.  f.  Bakteriol.  u.  Parasitenk.,  Jena,  1899, 
Ite  Abt,  Bd.  xxvi,  S.  548.] 

5  Ztschr.f.  Hyg.,  Leipzig,  1901,  Bd.  xxxvi,  S.  299. 


206  Chapter  VII 

The  facts  and  views  analysed  in  these  two  chapters  afford  us  a 
general  picture  of  the  phenomena  exhibited  in  natural  immunity 
against  micro-organisms.  The  dominant  feature  is  represented  by 
the  phagocytic  reaction  that  is  observed  throughout  the  animal 
series  and  that  is  exercised  against  parasites  belonging  to  all  the 
microbial  groups.  Phagocytosis  is  exhibited  not  only  by  the  macro- 
[2i7]phages  but  also,  in  a  high  degree,  by  the  microphages  which  stand 
out  as  the  defensive  cells  par  excellence  against  micro-organisms. 
Their  action  is  divided  into  a  series  of  vital  physiological  acts,  such 
as  sensitiveness  to  the  micro-organisms  and  their  products,  amoeboid 
movements  which  serve  to  ingest  the  micro-organisms,  and  into 
chemical  and  physico-chemical  processes,  such  as  the  destruction  and 
digestion  of  the  devoured  organisms. 

The  phagocytes  enter  into  a  struggle  against  the  micro-organisms 
and  rid  the  animal  organism  of  them  without  requiring  any  previous 
help  on  the  part  of  the  body  fluids.  Phagocytosis,  exercised  against 
living  and  virulent  micro-organisms,  is  sufficient  to  ensure  natural 
immunity.  The  bactericidal  power  of  the  serum,  which  for  a  long 
time  served  as  the  basis  for  a  humoral  theory  of  immunity,  represents 
merely  an  artificial  property,  developed  in  consequence  of  the  setting 
free  of  the  microcytase  of  the  leucocytes  that  have  become  disinte- 
grated after  the  blood  has  been  drawn.  The  agglutinative  power  of 
the  normal  fluids  of  the  body  plays  no  important  part  in  natural 
immunity. 

The  phagocytes,  in  order  to  fulfil  their  function,  can  attack 
micro-organisms  that  are  capable  of  producing  toxins.  Any  anti- 
toxic action  against  these  bacterial  poisons  is  in  no  way  necessary 
to  allow  of  phagocytosis  coming  into  action. 

Taken  as  a  whole,  the  data  collected  on  natural  immunity  against 
micro-organisms  clearly  demonstrate  that  the  destruction  of  these 
parasites  in  the  refractory  animal  organism  represents  merely  a  special 
phase  of  the  resorption  of  formed  elements. 


CHAPTER    VIII  [sis] 

SURVEY  OF  THE  FACTS  BEARING  ON  ACQUIRED 
IMMUNITY  AGAINST  MICRO-ORGANISMS 

The  discovery  of  attenuated  viruses  and  its  application  to  vaccination  against 
infective  diseases. — Vaccination  by  microbial  products. — Vaccination  with 
serums.— The  acquired  immunity  of  the  frog  against  pyocyanic  disease. — The 
acquired  immunity  against  vibrios. — Extracellular  destruction  of  the  cholera 
vibrio. — Part  played  by  two  substances  in  Pfeiffer's  phenomenon. — Specificity  of 
fixatives. — Phagolysis  and  its  relation  to  the  extracellular  destruction  of  vibrios. — 
Part  played  by  phagocytosis  in  the  acquired  immunity  against  vibrios. — Fate 
of  the  spirilla  of  recurrent  fever  in  the  organism  of  immunised  guinea-pigs. — 
Acquired  immunity  against  the  bacteria  of  typhoid  fever  and  pyocyanic  disease. 
— Acquired  immunity  against  swine  erysipelas  and  anthrax. — Acquired  immunity 
against  the  streptococcus. — The  acquired  immunity  of  rats  against  Trypanosoma. 

CERTAIN  of  the  hypotheses  on  acquired  immunity  are  of  as  ancient 
origin  as  are  those  on  natural  immunity.  For  example,  it  has  for  long 
been  known  that  man  is  constitutionally  refractory  to  certain  diseases 
which  are  very  fatal  to  cattle.  It  has  also  been  recognised  that  after 
a  first  attack  of  a  contagious  disease,  such  as  small-pox,  measles, 
scarlatina,  typhoid  fever,  etc.,  man  acquires  a  lasting  immunity ;  and 
that  the  same  rule  applies  to  domestic  animals,  for  example,  cattle 
that  have  recovered  from  cattle  plague  or  sheep  that  have  got  better 
from  sheep-pox,  become  refractory  against  these  diseases. 

The  discoveries  of  variolisation  and  vaccination,  as  methods  of 
conferring  on  man  a  resistance  to  small-pox,  have  notably  advanced 
our  knowledge  upon  acquired  immunity.  The  researches  on  the 
properties  of  vaccine  had  already  led  to  some  important  results. 
But  it  is  only  since  the  publication  of  Pasteur's  investigation,  carried 
out  with  his  collaborators  Chamber-land  and  Roux,  in  the  first  place, 
and  with  Thuillier  later,  that  we  have  been  able  to  take  up  the  study 
of  acquired  immunity  hi  a  really  scientific  manner.  The  first  in  this 


208  Chapter  VIII 

[219]  series  of  discoveries,  which  have  opened  up  a  path  so  fruitful  to 
science  and  medical  art,  was  the  discovery  of  the  attenuation  of 
micro-organisms.  The  small  cocco-bacillus  of  fowl-cholera  after 
several  weeks'  culture  in  broth  was  found  to  have  become  markedly 
attenuated  in  virulence.  To  Pasteur  the  idea  occurred  of  testing 
whether  fowls  that  had  resisted  the  inoculation  of  these  attenuated 
organisms  had  acquired  any  real  immunity  against  virulent  fowl- 
cholera.  Experiment  confirmed  his  expectation  and  led  to  the 
discovery  of  the  vaccine  against  this  disease.  The  method  was  at 
once  applied  to  other  infective  epizootic  diseases  and  shortly  after- 
wards Pasteur,  Chamberland  and  Roux  found  a  method  of  preserving 
sheep  and  cattle  from  anthrax.  To  attain  this  end  it  was  found 
necessary  to  prevent  the  bacillus  from  producing  spores  (in  this  they 
succeeded  by  cultivating  it  in  broth  at  a  temperature  of  42*5°  C.), 
because  these  spores  fix  the  virulence  and  prevent  attenuation.  Having 
overcome  this  main  obstacle,  Pasteur  and  his  collaborators  found 
that  their  cultures,  thus  deprived  of  spores,  become  attenuated  on 
exposure  to  the  air  and  so  become  transformed  into  vaccines.  They 
were  thus  able  to  prepare  their  two  anthrax  vaccines  which  soon 
found  such  wide  application  in  practice.  A  few  years  later,  Pasteur 
and  Thuillier  discovered  the  vaccines  against  swine  erysipelas  and, 
in  collaboration  with  Roux  and  Grancher,  Pasteur  made  the  first 
application  of  his  discoveries  to  the  vaccination  of  man  against 
rabies. 

The  path  thus  opened  up  was  traversed  by  many  other  investi- 
gators and  led  to  many  very  remarkable  discoveries.  Vaccination 
with  micro-organisms  became  a  recognised  method  and  in  the  hands 
of  Arloing,  Cornevin  and  Thomas,  soon  found  its  application  to 
symptomatic  anthrax.  The  next  step  in  this  onward  progress  of 
science  was  taken  when  Salmon  and  Smith,  working  at  hog- 
cholera,  demonstrated  the  possibility  of  vaccinating  not  only  with  hog- 
cholera  bacilli,  but  also  with  culture  fluids  in  which  these  organisms 
had  developed.  These  fluids,  when  completely  deprived  of  micro- 
organisms by  filtration,  protected  the  experimental  animals  from 
virulent  hog-cholera.  This  discovery,  at  first  sceptically  received, 
was  soon  confirmed  and  extended  by  the  work  of  other  investigators. 
Beumer  and  Peiper  extended  it  to  the  experimental  disease  set  up 
by  the  typhoid  bacillus  in  small  laboratory  animals  ;  Charrin  applied 
it  to  the  disease  that  he  produced  by  means  of  the  bacillus  of  blue 

[220]  pus  ;  and  Chamberland  and  Roux  prepared  vaccines  from  the  soluble 


Facts  bearing  on  acquired  immunity  209 

products  of  the  septic  vibrio  and  of  the  bacillus  of  symptomatic 
anthrax.  And  now,  as  the  result  of  these  investigations,  vaccinations 
by  microbial  products  are  in  everyday  use  in  all  research  laboratories. 
The  vaccinations  now  used  (anthrax,  symptomatic  anthrax,  swine 
erysipelas  and  rabies)  are  still  being  carried  out  by  means  of  the 
inoculation  of  living  viruses. 

The  comparative  history  of  acquired  immunity  is  still  very  in- 
complete. The  facts  known  concerning  the  adaptation  of  unicellular 
organisms  to  all  kinds  of  injurious  influences  of  a  physical  or  chemical 
nature  enable  us  to  perceive  that  acquired  immunity  is  just  as  general 
in  living  beings  as  is  natural  immunity ;  but  it  is  impossible,  in  the 
present  state  of  our  knowledge,  to  confirm  this  hypothesis  by  exact 
and  experimental  data.  The  reason  for  this  lies  in  the  great  difficulty 
we  have  in  carrying  out  experiments  on  the  lower  animals.  The 
majority  of  the  Invertebrata  in  captivity  do  not  remain  alive  long 
enough  and  can  not  be  sufficiently  often  inoculated  for  us  to  obtain 
in  them  a  well  marked  acquired  immunity  against  micro-organisms. 
Kowalevsky1,  the  celebrated  Russian  zoologist,  has  tried  to  overcome 
these  various  difficulties  by  making  use  of  Myriapods.  He  found  first 
that  Scolopendrae^  when  inoculated  with  anthrax  bacilli,  die  there- 
from during  the  heats  of  summer,  the  blood  containing  a  number 
of  anthrax  bacilli.  But  when  the  temperature  does  not  exceed  17° — 
18°  C.,  a  fairly  large  number  of  these  myriapods  survive.  The  same 
survival  was  observed  when  Pasteur's  first  vaccine  was  injected.  Kow- 
alevsky utilised  the  Scolopendrae  that  had  resisted  the  first  injection 
of  anthrax  bacilli  to  ascertain  whether  they  had  contracted  an  acquired 
immunity.  The  results  were  not  absolutely  demonstrative  and  Kow- 
alevsky sums  up  his  results  in  the  following  words :  "  I  cannot  say, 
therefore,  that  I  have  succeeded  in  solving  this  question  of  vaccina- 
tion, but  it  appears  to  me  very  probable  "  (p.  607). 

In  view  of  this  doubt,  I  asked  Mesnil  to  make  a  fresh  attempt, 
making  use  of  Scolopendrae  and  inoculating  them  with  anthrax 
bacilli.  These  creatures,  however,  were  so  delicate  and  so  little 
capable  of  remaining  alive  under  the  artificial  conditions  of  their 
captivity,  that  the  attempt  soon  had  to  be  abandoned.  I  tried  to 
obtain  better  results  with  the  larvae  of  Oryctes  nasicornis ;  here  [221] 
again  the  difficulties  were  too  great.  These  insects  exhibit  a 
perfect  natural  immunity  against  certain  micro-organisms,  but  for 
others  they  showed  an  insurmountable  susceptibility.  It  is  very 

1  Arch,  de  zool.  exper.,  Paris,  1895,  3fl  s6rie,  t.  m,  p.  591. 
B.  14 


210  Chapter  VIII 

evident,  then,  that  it  is  not  an  easy  matter  to  set  up  an  acquired 
immunity  in  the  Invertebrata. 

It  was  necessary,  therefore,  to  go  higher  up  the  animal  scale  and 
have  recourse  to  cold-blooded  vertebrates.  The  choice  naturally 
fell  on  the  frog.  I  asked  Dr  Gheorghiewski1,  who  was  working 
in  my  laboratory,  to  try  to  vaccinate  the  Batrachians  against  pyo- 
cyanic  disease.  I  ought  first  to  state  that  the  bacillus  of  blue  pus 
is  pathogenic  for  the  frog,  which  it  kills  both  at  the  ordinary  laboratory 
temperature,  and  at  that  of  the  incubator,  30°— 37°  C.  In  the  first 
case  the  fatal  dose  is  much  greater  than  in  the  second,  but  it  is 
always  easy  to  induce  a  fatal  infection.  In  this  respect,  therefore,  the 
Bacillus  pyocyaneus  is  much  better  adapted  for  study  than  the 
anthrax  bacillus  or  many  other  micro-organisms.  Gheorghiewski 
vaccinated  green  frogs  (Rana  esculenta),  which  had  been  accustomed 
to  the  incubator  temperature,  30°  C.,  by  injecting  every  4  to  7  days 
considerable  doses  of  cultures  of  Bacillus  pyocyaneus  heated  to  80°  C. 
in  order  to  kill  all  the  micro-organisms.  Some  (3 — 4)  weeks  after- 
wards the  prepared  frogs  became  more  resistant  to  the  Bacillus 
pyocyaneus  than  were  the  controls  placed  under  the  same  conditions. 
The  frogs,  inoculated  with  a  fatal  dose  of  the  bacilli,  clearly  exhibited 
a  certain,  though  slight,  degree  of  acquired  immunity.  They  withstood 
a  dose  that  was  always  fatal  to  the  controls  or  even  a  dose  and  a  half, 
but  died  when  injected  with  double  the  fatal  dose.  The  lymphatic 
fluid  of  the  vaccinated  frogs  feebly  agglutinated  (1  :  20 — 1  :  30)  the 
Bacillus  pyocyaneus  although  it  still  formed  an  excellent  culture 
medium  for  this  organism.  Gheorghiewski  satisfied  himself  that  the 
agglutination  was  insufficient  to  assure  immunity  to  the  frog.  The 
bacilli  agglutinated  into  clumps  were  very  virulent. 

A  detailed  examination  of  the  phenomena  observed  in  the  im- 
munised frogs  revealed  the  following  facts.  During  the  earliest 
stage  the  bacilli,  injected  into  the  dorsal  lymphatic  sac,  were  found 
free  in  the  fluid,  retained  their  form  and  were  not  transformed  into 
granules.  The  bacilli  carried  by  the  lymphatic  current  spread  rapidly 
[222]  throughout  the  body.  Very  shortly  after  inoculation,  however,  some 
of  the  leucocytes  began  to  ingest  the  bacilli  which  became  trans- 
formed into  spherules  within  these  cells.  Later,'  the  phagocytic 
reaction  increased  and  at  the  end  of  15  to  20  hours  all  the  bacilli 
were  found  inside  leucocytes.  It  was  easy  to  demonstrate  that  these 

1  Ann.  de  Vlnst.  Pasteur,  Paris,  1899,  t.  xm,  p.  314. 


Facts  bearing  on  acquired  immunity  211 

bacilli  had  been  ingested  in  a  living  condition.  Forty-eight  hours 
after  inoculation,  110  bacilli  were  to  be  found  in  the  lymph  of  the 
dorsal  sac,  either  inside  or  outside  the  cells.  But  this  fluid  when 
sown  on  nutrient  media  gave  colonies  of  the  Bacillus  pyocyaneus 
up  to  15  and  even  18  days  after  inoculation. 

We  may  conclude  from  these  facts  that  the  cold-blooded  verte- 
brata  are  capable  of  acquiring  immunity  to  a  slight  degree  and 
that,  in  this  acquired  immunity,  a  marked  phagocytosis  may  be  ob- 
served, but  no  bactericidal  action  of  the  fluids. 

In  order  to  gain  a  more  complete  idea  of  the  mechanism  of  ac- 
quired immunity,  it  is  necessary  to  observe  it  in  higher  vertebrates 
in  which  a  well  developed  immunity  of  this  type  is  readily  obtained. 
Here  we  must  have  recourse  to  mammals  and  pass  in  review  an  ample 
number  of  examples,  before  we  attempt  to  give  to  our  readers  a 
general  summary  of  the  question. 

For  long,  researches  on  acquired  immunity  were  confined  almost 
exclusively  to  the  analysis  of  the  facts  observed  in  animals  sub- 
mitted to  anti-anthrax  vaccinations  by  means  of  the  two  vaccines 
of  Pasteur.  A  large  number  of  important  facts  were  thus  collected, 
the  more  weighty  of  which  must  be  presented  to  the  reader.  But, 
before  entering  on  the  subject,  a  general  orientation  on  acquired 
immunity  in  laboratory  animals  against  vibrios  is  indispensable  as 
this  example  dominates,  so  to  speak,  the  whole  of  the  chapter  on 
acquired  immunity  against  micro-organisms. 

Von  Behring  and  Nissen1,  in  their  researches  on  the  bactericidal 
power  of  serums,  examined,  amongst  others,  several  specimens  of 
serums  coming  from  animals  that  had  been  vaccinated  against  various 
micro-organisms.  In  the  majority  of  the  examples  given  by  them  the 
acquired  immunity  produced  no  increase  in  this  power,  but  the  blood 
serum  of  guinea-pigs  that  had  been  immunised  against  Gamaleia's  vibrio 
(Vibrio  metchnikovi)  was  found  to  be  much  more  bactericidal  as  regards  [223] 
this  micro-organism  than  the  serum  of  normal  susceptible  guinea-pigs. 
These  authors  came  to  the  conclusion  that  in  acquired  immunity,  at 
least  as  regards  the  vibrio  mentioned,  the  chief  part  is  played  by  a 
bactericidal  substance  which  is  developed  in  the  fluids  of  the  vacci- 
nated animals.  They  were  content  with  the  mere  demonstration  of  this 
fact  without  making  any  attempt  to  follow  the  course  of  events  in  the 
destruction  of  the  vibrios  as  it  occurs  in  the  organism  of  the  vaccinated 

1  Ztschr.f.  Hyg.,  Leipzig,  1890,  Bd.  vui,  S.  412. 

14—2 


212  Chapter  VIII 

guinea-pig.  R.  Pfeiffer1  in  collaboration  with  Issaeff  sought  to  fill 
this  gap.  But,  instead  of  taking  Gamaleia's  vibrio,  these  observers 
concentrated  their  attention  on  the  study  of  the  acquired  immunity 
of  guinea-pigs  against  the  cholera  vibrio.  As  this  vibrio  is  as  a  rule 
less  virulent  than  Gamaleia's  vibrio,  it  was  necessary,  in  order  to 
obtain  a  fatal  infection,  to  inject  it,  not  into  the  subcutaneous  tissue 
but  into  the  peritoneal  cavity.  We  have  already  seen  (Chapter  VI) 
that  the  cholera  vibrio  when  inoculated  into  the  peritoneal  cavity  of 
the  guinea-pig,  there  meets  with  a  vigorous  resistance  on  the  part 
of  the  leucocytes  which  seize  the  living  and  virulent  vibrios  and 
digesting  them  rid  the  animal  of  their  presence.  But  when  the  dose 
of  the  vibrios  is  increased,  they  multiply  in  spite  of  the  phagocytic 
reaction ;  they  are  found  swarming  in  the  peritoneal  cavity,  whence 
they  invade  the  lymphatic  and  blood  vessels  and  cause  the  death 
of  the  animal.  It  is  easy,  then,  to  induce  a  fatal  infection  of  the 
guinea-pig  with  the  cholera  vibrio.  But  it  is  also  easy  to  vaccinate 
these  animals  against  this  experimental  disease.  We  have  only  to 
inoculate  them  with  a  non-fatal  quantity  of  living  cholera  vibrios, 
or  to  inject  into  them  a  culture  in  which  the  vibrios  have  been  killed 
by  heat,  or  some  of  the  culture  fluid  from  which  the  vibrios  have  been 
removed  by  filtration.  All  these  methods  soon  produce  an  acquired 
immunity  in  guinea-pigs.  If,  when  this  has  been  brought  about,  a 
little  blood  is  withdrawn  and  to  the  serum  a  small  quantity  of 
cholera  vibrios  is  added,  in  vitro,  we  can  readily  demonstrate  their 
disappearance,  under  the  influence  of  the  bactericidal  substance  dis- 
solved in  the  fluid.  In  this  respect  there  is,  then,  a  marked  analogy 
with  the  fact  established  by  v.  Behring  and  Nissen  as  regards 
Gamaleia's  vibrio. 

When  into  the  peritoneal  cavity  of  vaccinated  guinea-pigs  a 
certain  quantity  of  cholera  culture  containing  virulent  and  very 
[224]  motile  vibrios  is  injected,  we  find  that  in  the  peritoneal  fluid  drawn 
off  by  means  of  a  fine  pipette,  the  vibrios  have  undergone  profound 
changes  in  the  refractory  organism.  Even  a  few  minutes  after 
the  injection  of  the  vibrios,  the  leucocytes  disappear  almost  com- 
pletely from  the  peritoneal  fluid ;  and  only  a  few  small  lymphocytes 
and  a  large  number  of  vibrios,  the  majority  of  which  are  already 
transformed  into  granules,  are  found  (fig.  39);  and  there  is  pre- 
sented a  most  typical  case  of  Pfeiffer 's  phenomenon.  Alongside 

1  Ztschr.f.  Hyg.,  Leipzig,  1894,  Bd.  xvn,  S.  355,  and  Deutsche  med.  Wchnschr., 
Leipzig,  1896,  SS.  97,  119. 


Facts  bearing  on  acquired  immunity  213 

the  round  granules  may  be  seen  swollen  vibrios,  and  others  which 
have  kept  their  normal  form,  but  all  are  absolutely  motionless.   Some 
of  these  granules  are  gathered  into   small 
clumps,  others  remain  isolated  in  the  fluid. 
When  to  the  hanging  drop  containing  these 
transformed   vibrios    a   small    quantity   of  O%  ^ 

a  dilute  aqueous  solution  of  methylene  blue 
is  added,  we  observe  that  certain  granules  # 

stain    very  deeply,   whilst  others  take  on  ^  ^c 

merely  a  very  pale   tint,   scarcely  visible.  $ 

Many  of  these  granules  are  still  alive,  because 
it  is  easy  to  watch  them  develop  outside 
the  animal  and  elongate  into  new  vibrios. 
A  large  number  of  the  granules,  however, 

no  longer  exhibit  any  sign  of  life  and  are          showing  Pfeiflfer's  phe- 
evidently  dead.    R.  Pfeifler  and  certain  other         nomenon. 
observers  affirm   that  the  granules  may  be 

completely  dissolved  in  the  peritoneal  fluid  just  as  a  piece  of  sugar 
dissolves  in  water.  We  have  repeatedly  sought  for  this  disappearance 
of  the  granules  in  hanging  drops  of  the  peritoneal  fluid,  without 
being  able  to  find  any  diminution  in  the  number  of  these  trans- 
formed vibrios,  even  after  several  days ;  nor  have  we  been  able  to 
observe  the  phenomenon  of  the  solution  of  the  granules.  It  is  at  any 
rate  indisputable  that  this  granular  transformation  is  a  manifestation 
of  very  profound  lesions  undergone  by  the  cholera  vibrios  under  the 
influence  of  the  peritoneal  fluid  of  the  immunised  animal. 

An  attempt  has  been  made  to  define  the  mechanism  of  Pfeifler's 
phenomenon  more  exactly,  and  Fischer1  has  sought  to  refer  it  to 
osmotic  action,  exercised  by  the  salts  of  the  fluids  in  which  the  [225] 
vibrios  are  suspended.  These  vibrios,  under  the  action  of  media 
richer  or  poorer  in  salts  than  is  the  fluid  in  which  they  had  developed, 
are  said  to  present  an  increase  of  their  internal  pressure,  in  con- 
sequence of  which  the  vibrios  swell  up  or  allow  a  spherical  droplet 
of  protoplasm  to  escape  at  one  of  their  poles.  This  explanation  was 
inadequately  supported  by  its  author  and  cannot  be  regarded  as 
proved.  On  the  other  hand,  one  is  compelled  to  the  conclusion  that 
the  granular  transformation  is  due,  as  we  shall  see  later,  to  a  fermen- 
tative action  of  the  peritoneal  exudation. 

Whilst  the  vibrios  are  undergoing  this   transformation  in  the 
1  Zlschr.f.  Hyg.t  Leipzig,  1900,  Bd.  xxxv,  S.  1. 


214  Chapter  VIII 

peritoneal  cavity  of  an  immunised  guinea-pig,  the  animal  recovers 
from  a  malaise  that  is  quite  transitory  and  continues  to  live,  whilst 
normal  unvaccinated  guinea-pigs  die,  an  enormous  quantity  of  vibrios 
swarming  in  the  peritoneal  exudation.  The  difference  between  the  two 
animals  is  most  striking,  and  we  can  readily  understand  that  Pfeiffer 
was  so  impressed  by  it  that  he  was  led  to  attribute  the  acquired 
immunity  of  his  guinea-pigs  solely  to  the  granular  transformation 
set  up  by  a  bactericidal  substance  contained  in  the  fluids  of  the 
immunised  animals. 

The  ease  with  which  we  can  gain  an  idea  of  the  change  of  form 
in  the  vibrios  under  the  influence  of  the  fluids  of  the  body,  greatly 
aids  the  study  of  the  bactericidal  substance.  Before  passing  to  the 
question  of  the  part  played  by  this  substance  in  acquired  immunity 
we  must  consider  for  a  moment  the  principal  properties  of  this  acquired 
immunity.  Very  manifest  in  the  peritoneal  fluid,  the  power  of  causing 
Pfeiffer's  phenomenon  is  equally  evident  in  the  blood  serum  of  im- 
munised guinea-pigs,  as  has  been  demonstrated  by  Bordet.  A  drop 
of  this  serum,  when  quite  fresh,  readily  and  rapidly  transforms  a 
number  of  vibrios  into  granules.  When  the  serum  is  kept  for  several 
days  or  has  been  heated  to  55°  C.  for  an  hour,  the  total  disappearance 
of  the  substance  which  produces  Pfeiffer's  phenomenon  is  brought 
about.  This  at  once  betrays  the  presence  of  microcytase  in  the 
fluids  of  guinea-pigs  that  have  acquired  immunity  against  the  cholera 
vibrio.  Yet  the  blood  serum  and  the  peritoneal  fluid  of  these 
animals,  having  been  deprived  of  their  microcytase  by  heating 
to  55°  or  56°  C.,  still  retain  a  remarkable  power  over  the  vibrios. 
These  organisms  no  longer  undergo  granular  transformation,  under 
[226]  the  influence  of  the  heated  body  fluids,  but  they  are  deprived  of  all 
power  of  motion,  agglutinate  into  clumps  and  acquire  a  special 
susceptibility  to  the  action  of  cytase.  Soon  after  the  discovery  of 
Pfeiffer's  phenomenon,  I1  was  able  to  demonstrate  that  this  granular 
transformation  can  be  obtained  in  vitro  under  the  following  con- 
ditions. Prepare  a  hanging  drop  with  the  blood  serum  of  a  guinea- 
pig  vaccinated  against  the  cholera  vibrio,  a  serum  which  has  lost  the 
power  of  transforming,  by  itself,  the  vibrios  into  granules.  Add  to 
it  a  drop  of  the  peritoneal  lymph  of  a  normal  unvaccinated  guinea- 
pig;  this  lymph  contains  dead  or  living  leucocytes  and  is,  by  itself, 
also  incapable  of  producing  Pfeiffer's  phenomenon.  When,  to  the 
mixture  of  these  two  fluids,  which  are  inactive  when  they  are  employed 
1  Ann.  de  Vlnst.  Pasteur,  Paris,  1895,  t.  ix,  p.  433. 


Facts  bearing  on  acquired  immunity  215 

separately,  a  few  cholera  vibrios  are  added,  they  are  quickly  trans- 
formed into  granules.  This  transformation,  obtained  in  vitro,  is 
remarkably  like  that  produced  in  the  peritoneal  cavity  of  the  vac- 
cinated animal. 

Jules  Bordet1,  working  in  my  laboratory,  made  a  very  com- 
plete investigation  of  Pfeiffer's  phenomenon  outside  the  animal  body 
and  found  that,  in  my  experiment,  the  peritoneal  lymph  can  be 
replaced  by  the  blood  serum  of  the  vaccinated  guinea-pig  without 
thereby  in  the  least  hindering  the  granular  transformation.  After 
making  a  specially  thorough  study  of  the  question  he  has  come 
to  the  conclusion  that  Pfeiffer's  phenomenon  is  the  result  of  the 
action  of  two  substances.  One  of  these  is  found  in  the  blood  serum 
and  in  the  peritoneal  fluid  of  guinea-pigs  vaccinated  against  cholera, 
heated  to  55° — 56°  C.  or  deprived  by  some  other  means  of  their  indi- 
vidual power  of  transforming  vibrios  into  granules.  This  substance 
resists  this  temperature  and  only  loses  its  activity  on  being  heated 
to  68° — 70°  C.  The  second  of  the  two  substances,  that  found  in  the 
peritoneal  lymph  or  in  the  blood  serum  of  the  normal  guinea-pig, 
is,  on  the  other  hand,  destroyed  at  55° — 56°  C.  and  is  nothing  but  the 
ordinary  cytase  (or  alexine)  present  in  the  fluids  of  normal  animals. 

The  facts  we  have  described  with  regard  to  Pfeiffer's  phenomenon 
in  the  body  fluids  of  immunised  animals  must,  then,  be  interpreted 
as  follows.  The  fresh  peritoneal  exudation  or  blood  serum  of  these 
animals  readily  produces  the  granular  transformation,  because  in 
these  fluids  both  the  two  necessary  substances  are  found.  But  as 
microcytase  is  a  very  unstable  substance  which,  under  the  influence  [227] 
of  time  or  heating  to  55° — 56°  C.,  is  destroyed,  the  fluids  of  im- 
munised animals  are  very  readily  deprived  of  it.  The  blood  serum 
then,  after  being  some  time  outside  the  body,  becomes  incapable  of 
transforming  vibrios  into  granules ;  but  when  to  it  is  added  a  small 
quantity  of  the  cytase,  found  in  the  blood  serum  or  in  the  peritoneal 
lymph  of  the  normal  guinea-pig,  the  transformation  takes  place  with 
great  rapidity.  To  the  serum  of  the  immunised  animal,  which  has 
become  inactive,  is  restored  its  property  of  setting  up  Pfeiffer's 
phenomenon.  This  interpretation,  formulated  by  Bordet,  corresponds 
to  the  whole  of  the  known  data  and  is  now  generally  accepted. 

As  the  fluids  of  immunised  animals,  that  have  become  incapable 
of  transforming  vibrios  into  granules,  still  retain  their  power  of 
rendering    these    organisms  motionless   and  of   uniting  them  into 
1  Ann.  de  Vlnst.  Pasteur,  Paris,  1895,  t.  IX,  p.  462. 


216  Chapter  VIII 

clumps,  it  might  be  asked  whether  this  agglutinative  substance  might 
not  be  the  substance,  stable  under  heat,  which  is  necessary  for  the 
production  of  Pfeiffer's  phenomenon.  For  some  time,  indeed,  it  was 
believed  that  this  phenomenon  is  due  to  the  microcytase  acting  on 
vibrios  which  have  first  been  modified  by  the  agglutinative  sub- 
stance. This  latter  substance  resists  heating  to  55° — 56°  C.,  is  only 
destroyed  at  higher  temperatures,  and  is  retained  in  the  blood  serum 
long  after  the  cytase  has  entirely  disappeared.  The  analogy  between 
the  agglutinative  substance  of  the  fluids  of  animals  that  have  acquired 
immunity  and  the  substance  in  the  same  fluids  that  is  stable  under 
heat  is  undeniable,  and  yet  these  two  substances  are  not  identical. 
A  whole  series  of  observations,  which  we  shall  presently  describe, 
demonstrate  this  thesis  clearly.  A  serum  may  be  highly  agglutinative 
without  being  capable  of  bringing  about  the  transformation  of  vibrios 
into  granules ;  the  converse  also  holds  good.  The  substance  which  sets 
up  Pfeiffer's  phenomenon,  and  which  is  found  in  the  fluids  of  immunised 
guinea-pigs,  is  a  "fixative  substance"  analogous  to  those  we  have 
already  met  with  in  the  serums  of  animals  so  adapted  that  they  are 
able  to  resorb  the  various  cell  elements.  As  in  the  resorption  of  cells, 
so  also  in  the  destruction  of  micro-organisms,  the  fixatives  are  specific. 
The  substance  which  aids  the  transformation  into  granules  is  not  only 
distinct  from  the  fixatives  which  sensibilise  red  blood  corpuscles  or 
spermatozoa,  but  also  from  the  fixatives  which  sensibilise  bacteria. 
This  specificity  has  been  demonstrated  and  carefully  studied  by 
Pfeiffer,  who  has  shown  that  it  may  even  serve  to  distinguish  species 
[228]  of  bacteria.  The  serum  of  a  guinea-pig  which  has  been  immunised 
against  the  cholera  vibrio,  will  render  sensitive  these  vibrios,  and 
these  only,  to  the  action  of  the  microcytase.  Even  allied  vibrios, 
such  as  various  water  vibrios,  for  example,  are  not  sensitive  to  the 
fixative  of  anticholera  serum.  On  the  other  hand,  the  serums  ob- 
tained after  the  inoculation  of  these  aquatic  vibrios  are  incapable  of 
producing  granular  transformation  in  the  cholera  vibrio. 

When  we  inject  into  one  and  the  same  animal  several  species 
of  vibrios  we  obtain  a  serum  or  a  peritoneal  fluid  which  produces 
Pfeiffer's  phenomenon  with  the  vibrios  of  all  the  species  which  have 
been  used  to  make  the  inoculations.  This  anti vibrio  serum  contains 
only  a  single  cytase  for  the  vibrios,  but  contains  as  many  different 
fixatives  as  there  were  species  inoculated. 

The  transformation  of  vibrios  into  granules,  when  produced  in  a 
high  degree  against  virulent  vibrios,  under  the  influence  of  the  body 


Facts  bearing  on  acquired  immunity  217 

fluids  of  immunised  animals,  affords  a  very  valuable  indication  of  the 
simultaneous  presence  of  cytase  and  of  specific  fixative.  As  we  have 
already  stated,  at  the  commencement  of  this  account  of  the  acquired 
immunity  of  guinea-pigs  against  the  cholera  vibrio,  Pfeiffer's  pheno- 
menon is  manifested  in  the  peritoneal  fluid  of  these  animals  in  a 
very  short  time  (5  to  20  minutes)  after  the  inoculation  of  the  vibrios. 
This  proves  that  in  this  fluid  the  two  substances  really  occur  together, 
and  that  the  fixative  and  the  cytase  are  in  solution  in  the  plasma  of 
the  exudation.  Is  it  the  same  in  every  part  of  the  body  of  immunised 
guinea-pigs  ?  If,  instead  of  introducing  the  cholera  vibrio  into  the 
peritoneal  cavity,  we  inject  it  into  the  subcutaneous  tissue  or  into  the 
anterior  chamber  of  the  eye  of  these  animals,  Pfeiffer's  phenomenon 
does  not  make  its  appearance.  The  vibrios,  isolated  or  collected  into 
small  clumps,  do  not  undergo  granular  transformation ;  they  keep 
their  normal  vibrio  form  and  remain  alive  much  longer  than  in  the 
peritoneal  cavity.  Some  of  them  may  be  found  still  living  24  hours 
after  subcutaneous  injection  and  several  (4 — 6)  days  in  the  anterior 
chamber  of  the  eye.  Xor  can  Pfeiffer's  phenomenon  be  observed  when 
the  cholera  vibrio  is  introduced  into  the  oedema  of  the  foot,  pro- 
duced in  consequence  of  the  slowing  of  the  circulation,  the  vibrios 
remaining  alive  for  a  fairly  long  time.  These  facts  clearly  indicate 
that  in  the  fluid  thrown  out  in  passive  oedema,  just  as  in  the  aqueous 
humour  of  the  eye  or  in  the  subcutaneous  tissue,  the  two  substances 
necessary  to  set  up  the  granular  transformation  are  not  present.  Are 
both  of  them  absent  or  only  one  ?  This  question  is  easily  answered  [229] 
on  adding  to  the  fluids  mentioned  normal  guinea-pig's  serum,  a 
serum  which,  by  itself,  is  incapable  of  producing  Pfeiffer's  pheno- 
menon. Bordet1  has  made  these  experiments  and  found  that  when 
to  the  fluid  of  the  passive  oedema  of  the  immunised  guinea-pig 
normal  serum  is  added,  this  fluid  transforms  the  cholera  vibrio  into 
granules,  but  does  so  in  less  degree  than  does  the  serum  of  the  same 
immunised  guinea-pig,  when  heated  to  55° — 56°  C.,  to  which  normal 
serum  has  likewise  been  added.  There  is,  then,  reason  to  conclude 
that  the  fluid  of  the  oedema  does  not  contain  any  cytase,  but  contains 
a  certain  quantity  of  cholera  fixative,  less,  however,  than  that  which 
is  found  in  the  blood  serum.  As  to  the  aqueous  humour  of  the  eye 
of  immunised  animals,  analogous  experiments  have  demonstrated 
that  it  contains  neither  of  the  two  substances  necessary  for  the 
production  of  Pfeiffer's  phenomenon. 

1  "Contribution  k  1'etude  du  serum  chez  les  animaux  vaccines,"  Ann.  Soc.  d.  sc. 
nat.  et  med.  de  Bruxelles,  1895,  t.  iv. 


218  Chapter  VIII 

With  the  help  of  the  facts  I  have  here  summarised,  we  arrive  at 
the  following  conclusion.  In  the  animal  that  is  immunised  against 
the  cholera  vibrio,  microcytase  is  found  in  the  peritoneal  exudation ; 
it  does  not  pass,  however,  either  into  the  fluid  of  the  passive  oedema 
or  into  the  aqueous  humour  of  the  eye ;  the  cholera  fixative  is 
found  in  the  peritoneal  fluid  and  passes  into  the  oedema,  but 
does  not  penetrate  into  the  fluid  of  the  eye.  This  indicates  that 
microcytase  is  found  in  fluids  rich  in  leucocytes,  but  is  absent  from 
those  which  contain  very  few  or  none  of  these  cells. 

The  introduction  of  vibrios  into  the  peritoneal  cavity  of  immunised 
guinea-pigs  at  once  produces  Pfeiffer's  phenomenon,  and  at  the  same 
time  causes  the  disappearance  of  the  majority  of  the  leucocytes  from 
the  peritoneal  lymph.  We  have  already  had  occasion,  several  times, 
to  speak  of  this  phagolysis,  because  it  is  produced  as  a  sequel  to  the 
injection  into  the  peritoneal  cavity  of  blood,  spermatic  fluid,  and  all 
kinds  of  fluids.  The  greater  the  quantity  of  fluid  injected  and  the 
greater  the  difference  of  the  temperature  between  it  and  the  contents 
of  the  normal  peritoneum  the  more  vigorous  is  phagolysis. 

Pierallini1,  working  in  my  laboratory,  studying  phagolysis  in  the 
peritoneal  cavity  of  the  guinea-pig,  has  obtained  several  results 
worthy  of  attention.  Of  all  the  fluids  used  by  him,  such  as  water, 
broth,  filtered  cultures  of  micro-organisms  and  physiological  saline 
[230]  solution,  the  last  of  these  caused  the  least  intense  phagolysis,  yet 
one  sufficiently  well  marked.  Immediately  after  the  injection  of  any 
of  the  above  fluids  the  number  of  leucocytes  in  the  peritoneal  lymph 
diminishes  very  considerably,  the  cells  being  found  collected  in 
clumps  on  the  omentum.  Many  of  them  exhibit  signs  of  enfeeble- 
ment  and  of  partial  destruction.  Alongside  the  leucocytes  are  found 
fibrinous  masses,  this  affording  evidence  that  some  of  the  leucocytes 
have  been  greatly  damaged  and  have  given  up  the  fibrin-ferment 
which  induces  coagulation  of  the  fibrin.  When  Pierallini  injected 
fluids  containing  coloured  powders  in  suspension,  such  as  Indian  ink 
and  vermilion,  he  observed  that  these  substances  accumulated  on  the 
greater  omentum,  which  became  stained  black  or  red.  Microscopical 
examination  revealed  the  existence  of  a  not  very  intense  phagocytosis 
and  a  number  of  free  coloured  granules  in  the  midst  of  filaments  of 
fibrin. 

The  leucocytes  which,  during  this  phagolysis,  allowed  the  fibrin- 
ferment  to  escape  might  also  give  up  a  certain  amount  of  their 
microcytase.    This  microcytase  would  pass  into  the  peritoneal  fluid 
1  Ann.  de  VInst.  Pasteur,  Paris,  1897,  t.  xi,  p.  308. 


Facts  bearing  on  acquired  immunity  219 

and  give  rise  to  Pfeiffer's  phenomenon.  If  this  hypothesis  be  correct, 
the  suppression  of  phagolysis  would  result  in  the  absence  of  the  trans- 
formation of  the  vibrios  into  granules.  It  is  not  a  difficult  matter 
to  verify  this  hypothesis  as  we  have  a  means  of  preventing  phago- 
lysis or  at  least  of  reducing  it  very  considerably.  Issaeff1,  in  an 
investigation  carried  out  in  Pfeifier's  laboratory,  demonstrated  that 
an  intraperitoneal  injection  of  physiological  salt  solution,  broth, 
urine,  etc.,  reinforces  the  leucocytes  and  brings  them  up  in  large 
numbers  into  the  peritoneal  cavity.  It  is  easy  to  foresee  that  such 
an  injection  would  serve  to  diminish  the  intensity  of  the  phagolysis. 
In  fact,  if  we  first  inject  a  few  cubic  centimetres  of  physiological  salt 
solution  or  of  fresh  broth  into  the  peritoneal  cavity  of  a  guinea-pig, 
and  if,  on  the  following  day,  we  repeat  the  same  operation,  we  shall 
find  that  after  the  second  injection  phagolysis  is  much  less  powerful 
than  after  the  first.  Pierallini,  who  repeated  these  experiments, 
observed  that  the  phagocytosis  of  the  coloured  granules  is  much 
more  complete  in  the  guinea-pigs  that  were  treated  by  a  preliminary 
injection  into  the  peritoneal  cavity.  The  amount  of  fibrin  on  the 
omentum  is  in  this  case  much  less,  and  the  phenomena  as  a  whole 
show  that  in  these  guinea-pigs  the  damage  to  the  leucocytes  is  very  [231] 
considerably  attenuated. 

We  have  been  able  to  demonstrate  that  in  the  case  where  phago- 
lysis is  thus  diminished,  Pfeiffer's  phenomenon  is  not  produced  or  is  • 
manifested  in  a  very  feeble  degree.  If  the  experiment  succeeds,  the 
fluid  taken  from  the  peritoneal  cavity  of  a  guinea-pig  prepared  the 
day  before  and  then  injected  with  a  culture  of  cholera,  is  opaque 
and  thick,  like  pus.  It  contains  a  mass  of  leucocytes  in  good  con- 
dition, a  large  number  of  which  gorge  themselves  in  a  few  minutes 
with  a  number  of  vibrios.  The  plasma  of  this  exudation  contains 
few  vibrios,  and  these  retain  their  normal  form  and  do  not  exhibit, 
save  exceptionally,  a  granular  change.  A  little  later  there  remain 
no  free  vibrios  ;  they  are  all  contained  within  leucocytes.  Pfeiffer2 
declared  himself  against  the  facts  I  have  just  summarised,  because  he 
was  never  able  to  prevent  the  granular  transformation  of  the  vibrios, 
in  spite  of  the  preparatory  injection  of  sodium  chloride.  Abel3,  who 
repeated  the  experiments,  expressed  an  intermediate  view:  in  guinea- 
pigs  prepared  by  injections  the  day  before,  he  observed  that  one 

1  Ztschr.f.  Hyg.,  Leipzig,  1894,  Bd.  xvi,  S.  287. 

2  Deutsche  med.  Wchnschr.,  Leipzig,  1896,  S.  120. 

3  Centralll.  f.  Bakteriol  u.  Parasitenk.,  Ite  Abt,  Jena,  1896,  Bd.  xx,  S.  761. 


220  Chapter  VIII 

portion  of  the  vibrios  became  transformed  into  granules,  whilst 
another  became  the  prey  of  the  leucocytes.  The  fact  is,  the  sup- 
pression of  phagolysis  demands  special  conditions  :  the  broth  that  is 
injected  must  be  freshly  prepared,  and  before  its  introduction  into 
the  peritoneal  cavity  it  must  be  heated  to  37° — 39°  C.  Even  when 
these  precautions  are  taken  it  sometimes  happens  that  the  experi- 
ment is  not  very  successful.  In  making  the  experiment  we  must  be 
guided  by  the  appearance  of  the  peritoneal  fluid  withdrawn  into 
the  small  glass  pipettes.  If  the  fluid  which  enters  the  tube  is  clear 
or  scarcely  clouded,  it  indicates  that  phagolysis  has  taken  place,  in 
spite  of  the  preparatory  injection.  The  experiment  is  successful 
in  those  cases  where  the  peritoneal  exudation  is  very  cloudy  and 
resembles  pus. 

As  the  demonstration  of  the  suppression  of  Pfeifler's  phenomenon 
as  well  as  that  of  phagolysis  is  of  fundamental  importance,  I  asked 
M.  Gamier1  to  carry  out  further  experiments  with  the  object  of 
setting  the  question  at  rest.  Using  a  whole  series  of  fluids  for 
the  preparatory  injection  he  found  that  fresh  broth  gives  the  best 
[232]  results.  In  guinea-pigs  in  which  the  phagolysis  had  been  reduced 
to  a  minimum,  phagocytosis  commenced  immediately  after  the  in- 
jection of  the  vibrios.  In  from  two  to  five  minutes  many  vibrios 
are  found  inside  the  leucocytes,  the  free  vibrios  now  being  few 
in  number  and  not  exhibiting  Pfeifler's  phenomenon.  Gamier  in 
his  memoir  gives  photographic  reproductions  of  leucocytes  crammed 
with  vibrios;  these  should  convince  the  veriest  sceptic.  Since  the 
publication  of  this  paper  no  objection  has  been  advanced,  and  this 
question  of  the  suppression  of  the  granular  transformation  of  vibrios 
may  now  be  regarded  as  definitely  settled.  I  have  since  demonstrated 
this  feature  to  many  observers,  all  of  whom  have  assured  themselves 
of  its  accuracy.  It  must,  then,  be  accepted  that  Pfeiffer's  phenomenon 
is  not  produced  in  the  peritoneal  cavity  except  when  there  is  phago- 
lysis. As  this  fact  renders  it  very  probable  that  the  microcytase, 
which  is  necessary  for  the  transformation  of  the  vibrios,  escapes  from 
the  injured  leucocytes,  it  becomes  necessary  to  verify  this  hypothesis 
by  a  series  of  other  experiments.  If  this  hypothesis  be  well  founded, 
Pfeiffer's  phenomenon  should  not  be  observed  in  those  situations  in 
the  body  where  there  are  no,  or  almost  no,  leucocytes  already  present. 
These  conditions  can  be  realised  by  injecting  cholera  vibrios  into  the 
subcutaneous  tissue  or  into  the  anterior  chamber  of  the  eye  of  guinea- 
1  Ann.  de  VInst.  Pasteur,  Paris,  1897,  t.  xi,  p.  767. 


Facts  'bearing  on  acquired  immunity  221 

pigs  that  are  well  vaccinated  against  the  cholera  vibrio.  Under  these 
conditions,  as  I  have  demonstrated  in  my  work  on  the  extracellular 
destruction  of  cholera  vibrios,  the  vibrios  retain  their  normal  form 
and  are  never  transformed  into  granules.  Pfeiffer  has  questioned 
this  result,  stating  that  beneath  the  skin  of  vaccinated  guinea-pigs 
the  granular  transformation  is  always  produced,  though  in  a  more 
feeble  fashion  and  after  more  delay  than  in  the  peritoneal  cavity. 
The  contradiction  between  Pfeiffer's  experiments  and  my  own  can, 
however,  be  explained.  When  inoculating  the  vibrios  into  the  sub- 
cutaneous tissue,  or  during  the  withdrawal  of  the  exudation  formed 
at  the  point  of  infection,  small  haemorrhages  are  sometimes  produced 
and  a  certain  amount  of  microcytase  is  set  free  from  the  leucocytes 
found  in  the  effusion  of  blood  ;  these  cells  also  give  up  to  the  extra- 
vasated  blood  a  portion  of  their  fibrin-ferment.  When  the  experi- 
ment is  successful,  that  is  to  say  when  no  haemorrhage  is  produced 
during  the  operations  involved,  the  subcutaneous  exudation  contains 
normal  vibrios  only,  Avithout  the  appearance  of  any  trace  of  Pfeiffer's 
phenomenon  in  the  fluid. 

If  the  extracellular  transformation  of  the  vibrios  into  granules  [233] 
were  the  real  cause  of  the  acquired  immunity,  the  absence  of  this 
phenomenon  in  the  subcutaneous  tissue  of  the  vaccinated  guinea- 
pig  should  lead  to  the  death  of  the  animal.  As  a  matter  of  fact 
this  does  not  take  place  and  the  animal  resists  the  inoculation  of  the 
vibrios.  This  conclusion  is  open  to  one  serious  objection.  As  the 
cholera  vibrio  in  the  great  majority  of  cases  is  incapable  of  producing 
a  fatal  infection  when  inoculated  subcutaneously,  even  in  normal 
unvaccinated  guinea-pigs,  this  example  of  immunity  must  be  placed 
in  the  category  of  natural  immunity,  a  kind  of  immunity  which  may  de- 
pend on  causes  other  than  those  on  which  acquired  immunity  depends. 
To  answer  this  objection  it  was  necessary  to  select  a  race  of  vibrios 
capable,  when  injected  subcutaneously,  of  causing  death.  Mesnil1,  chief 
of  my  laboratory  staff,  undertook  to  carry  out  experiments  with  the 
•Massowali  vibrio,  which  is  regarded  by  some  authors  as  belonging 
to  the  true  cholera  species.  When  inoculated  subcutaneously  into 
unprotected  guinea-pigs,  it  induces  local  oedema,  in  which  the  vibrios 
swarm ;  the  infection  rapidly  becomes  generalised  and  causes  the  death 
of  the  animal  in  24  hours.  Yet  this  vibrio,  when  injected  into  the 
subcutaneous  tissue  of  well  vaccinated  guinea-pigs,  is  completely 
resisted  by  these  animals  and  not  the  least  trace  of  Pfeiffer's  phe- 
1  Ann.  de  VInst.  Pasteur,  Paris,  1896,  t,  x,  p.  375. 


222  Chapter  VIII 

nomenon  is  produced.  Under  these  conditions,  a  certain  number  of 
the  vibrios  are  at  first  united  into  masses,  but  a  considerable  number 
remain  isolated  and  motile.  Some  hours  after  inoculation  the  number 
of  clumps  diminishes  and  the  isolated  vibrios  become  more  numerous, 
a  fact  which  indicates  a  certain  amount  of  adaptation  of  the  vibrio 
to  the  medium  in  which  it  now  finds  itself.  But  never,  so  long  as  the 
vibrios  remain  free  in  the  subcutaneous  exudation,  do  they  become 
transformed  into  granules. 

Salimbeni1,  in  an  investigation  carried  out  in  my  laboratory, 
endeavoured  to  satisfy  himself  whether  or  no  Pfeiffer's  phenomenon 
is  produced  in  the  subcutaneous  tissue  of  a  horse  that  had  been 
hyperimmunised  against  the  cholera  vibrio.  This  animal  had,  during 
a  period  of  1 4  months,  received  considerable  quantities  of  these  micro- 
organisms, and  the  serum  of  its  blood  transformed  the  vibrios  into 
granules  with  great  rapidity  and  intensity.  In  spite  of  such  favour- 
able conditions  for  the  manifestation  of  Pfeiffer's  phenomenon,  it 
was  never  produced  beneath  the  skin  of  this  horse.  The  vibrios 
when  injected  in  this  position  became  completely  motionless  in  a 
[234]  very  short  time,  but  they  kept  their  vibrio  form  and  remained  alive 
for  a  number  of  hours.  The  exudation  drawn  off  up  to  24  hours 
after  inoculation  still  gave  growths  of  the  cholera  vibrio. 

As  it  is  more  easy  to  introduce,  without  effusion  of  blood,  the 
cholera  vibrio  into  the  anterior  chamber  of  the  eye  than  beneath 
the  skin,  and  as  the  aqueous  humour  contains  no  fixative,  the  absence 
of  the  granular  transformation  in  the  first  of  these  two  situations  has 
been  observed  even  by  Pfeiffer  himself.  The  demonstration  of  this 
fact  presents  no  difficulty,  and  for  a  considerable  period  we  may 
observe  free  and  perfectly  motile  vibrios  moving  about  in  the  aqueous 
humour.  The  exudation  from  the  eye  contains  many  of  these  living 
organisms,  which  when  sown  on  culture  media  made  their  appearance 
as  colonies  even  when  the  fluid  has  been  withdrawn  from  the  eye 
several  days  after  inoculation. 

These  carefully  established  facts  show  very  clearly  that  the  micro- 
cytase  is  only  met  with  in  the  fluids  of  the  living  animal  in  those 
situations  in  which  there  are  many  pre-existing  leucocytes  and  under 
conditions  in  which  the  cells  undergo  a  more  or  less  marked  phago- 
lysis.  This  may  be  corroborated  by  a  decisive  experiment.  When 
we  inject  a  suspension  of  the  cholera  vibrio  directly  into  the  veins  of 
a  guinea-pig,  well  vaccinated  against  these  organisms,  and  whose 
de.  I'Inst.  Pasteur,  Paris,  1898,  t.  xn,  p.  199. 


Facts  hearing  on  acquired  immunity  223 

serum  produces  in  vitro  Pfeiffer's  phenomenon  with  great  rapidity, 
this  phenomenon  is  not  manifested.  This  experiment  has  been 
performed  and  described  by  Bordet1.  Having  injected  a  suspen- 
sion of  this  vibrio  into  the  jugular  vein  of  a  guinea-pig  vaccinated 
against  the  cholera  vibrio,  he  killed  the  animal  an  hour  later  and 
found,  in  the  blood  of  the  heart,  vibrios  that  had  kept  intact  their 
form  and  their  property  of  staining  with  methylene  blue.  Cultiva- 
tion of  the  blood  of  the  heart,  liver  and  spleen  gave  growths  of 
vibrios.  In  another  guinea-pig,  hypervaccinated  against  the  same 
organism  and  inoculated  by  the  same  method,  the  blood  drawn  off 
shortly  (4 — 15  minutes)  afterwards  showed,  in  preparations  treated 
with  methylene  blue,  well-stained  vibrios,  of  normal  form  and  quite 
intact.  This  is  the  most  direct  proof  of  the  absence  of  Pfeiffer's 
phenomenon  in  the  blood  fluid  of  a  living  animal  that  enjoys  a  very  [235] 
marked  acquired  immunity.  The  intact  vibrios  were  lodged  inside 
the  leucocytes. 

Levaditi2  repeated  these  experiments  in  my  laboratory  and  varied 
the  conditions  under  which  the  vibrios  were  injected  into  the  blood 
vessels.  He  was  sometimes  able  to  observe  phagolysis  of  the  leuco- 
cytes of  the  blood  and  their  almost  complete  disappearance  from  the 
the  peripheral  circulation.  In  these  cases  the  injured  leucocytes 
accumulated  in  the  pulmonary  capillaries  and  masses  of  them  were 
seen  surrounding  groups  of  vibrios  that  were  transformed  into 
granules.  It  was,  however,  easy  to  exclude  phagolysis  by  preparing 
the  animals  by  means  of  injections  of  physiological  saline  solution  or 
broth.  Under  these  conditions  the  leucocytes  remained  in  the  blood 
current  and  very  soon  ingested  the  vibrios.  Whilst  the  vibrios  that 
were  still  free  in  the  blood  plasma  retained  their  form  and  staining 
power  intact,  those  found  inside  microphages  were  already,  in  great 
part,  transformed  into  granules.  The  rapidity  with  which  these 
phagocytes  ingest  the  vibrios  and  set  up  the  changes  in  them  is  really 
extraordinary. 

In  this  case,  which  affords  a  typical  example  of  the  reaction  of  the 
animal  organism  in  acquired  immunity,  we  see  a  very  marked  and 
immediate  phagocytosis.  It  is  this  same  process  that  has  already 
been  described  as  occurring  in  the  peritoneal  cavity  of  vaccinated 
guinea-pigs  in  which  phagolysis  was  absent  as  the  result  of  pre- 
paratory injection.  In  the  subcutaneous  tissue  and  in  the  anterior 

1  Ann.  Soc.  d.  sc.  med.  et  nat.  de  Bruxelles,  1895,  t.  iv. 
3  Ann.  de  tlnst.  Pasteur,  Paris,  1901,  t  xv,  p.  894. 


224  Chapter  VIII 

chamber  of  the  eye,  where  Pfeiffer's  phenomenon  is  regularly  absent, 
the  phagocytosis  follows  its  ordinary  course  and  causes  the  destruction 
of  the  vibrios.  This  result  has  been  confirmed  repeatedly — see  works 
by  Bordet,  Mesnil  and  Salimbeni  already  quoted. 

We  need  only  compare  the  extension  of  Pfeiffer's  phenomenon 
and  that  of  phagocytosis  in  animals  that  are  immunised  against  the 
cholera  vibrio,  to  satisfy  ourselves  that  the  former  phenomenon  is  a 
limited  one  whilst  the  latter  is  general.  There  might  be  advanced 
against  the  latter  conclusion  the  fact  of  the  absence  of  any  ingestion 
of  the  vibrios  in  the  peritoneal  fluid  of  guinea-pigs  that  are  im- 
[236]  munised  but  are  not  preserved  against  phagolysis.  When  a  little  of 
the  peritoneal  fluid  is  drawn  off  with  small  tubes  shortly  after  the 
injection  of  vibrios  into  the  peritoneal  cavity,  as  a  matter  of  fact,  only 
a  very  intense  Pfeiffer's  phenomenon  is  seen,  phagocytosis  being  com- 
pletely or  almost  entirely  absent.  But  this  procedure  is  insufficient. 
If  we  are  to  get  an  idea  of  what  really  takes  place  in  the  abdominal 
cavity,  the  animal  must  be  killed  and  the  peritoneum  and  especially 
the  omentum  very  carefully  examined.  As  first  demonstrated  by 
Max  Gruber1  and  later  by  Cantacuzene2,  the  greater  omentum  is,  in 
these  cases,  covered  with  a  thick  layer  which  contains  a  large  number 
of  leucocytes,  of  which  some  are  filled  with  vibrios;  further,  this 
layer  contains  a  mass  of  vibrios,  in  part  transformed  into  granules, 
in  part  agglutinated  or  isolated  and  retaining  their  vibrionic  form 
intact  As  time  goes  on,  the  phagocytosis  becomes  more  and  more 
marked,  and  it  is  impossible  to  deny  its  existence  or  to  attribute  to 
it  merely  a  secondary  part. 

We  have  seen  that  the  suppression  of  Pfeiffer's  phenomenon  in 
the  peritoneal  cavity  and  in  the  blood,  or  its  total  absence  in  the 
anterior  chamber  of  the  eye,  does  not  in  the  least  deprive  the  vac- 
cinated guinea-pig  of  its  acquired  immunity.  The  animal  resists 
the  vibrios  perfectly,  without  these  requiring  to  be  transformed  into 
granules  in  the  body  fluids.  This  transformation  does  take  place 
undoubtedly,  but  only  inside  the  phagocytes.  As  already  stated  in 
the  discussion  on  natural  immunity  (Chaps.  VI,  VII)  the  vibrios, 
after  being  ingested  by  the  microphages,  almost  immediately  undergo 
within  these  cells  a  change  in  form,  very  similar  to  that  observed  in 
the  real  Pfeiffer's  phenomenon.  The  microphages  are  often  full  of 
a  quantity  of  granules,  derived  from  the  ingested  vibrios,  which  in 

1  Munchen.  med.  Wchnschr.,  1896,  SS.  277  and  310. 

2  Ann.  de  I'Inst.  Pasteur,  Paris,  1898,  t.  xn,  p.  273. 


Facts  bearing  on  acquired  immunity  225 

a  short  time  are  completely  digested.  This  fact,  of  such  constant 
occurrence  in  the  phagocytosis  of  the  vibrios,  furnishes  us  with  still 
another  proof  of  the  microphagic  origin  of  microcytase. 

If  Pfeiffer's  phenomenon  is  merely  a  particular  instance  of  the  trans- 
formation of  vibrios  into  granules  in  fluids  containing  microcytase, 
it  is  quite  natural  that  its  suppression  should  not  involve  a  fatal 
infection  of  the  vaccinated  animals.  On  the  other  hand,  if  the  phago- 
cytic  reaction,  so  widely  different,  really  plays  an  important  part 
in  acquired  immunity,  everything  that  interferes  with  phagocytosis 
must  at  the  same  time  compromise  the  refractory  condition.  With  [237] 
the  object  of  solving  this  question  Cantacuzene  \  working  in  my 
laboratory,  undertook  a  detailed  investigation  of  this  point.  He 
showed  that  the  injection  of  opium,  in  a  non-fatal  dose,  narcotised 
the  guinea-pig  and  at  the  same  time  prevented  the  movements  of 
the  leucocytes.  Small  glass  tubes  containing  cholera  vibrios  and 
introduced  beneath  the  skin  of  vaccinated  guinea-pigs,  became  filled 
with  numbers  of  leucocytes  in  the  non-narcotised  animal;  in  the 
guinea-pig  that  had  received  tincture  of  opium,  the  tubes  left  for 
several  hours  contained  no  leucocytes  and  later  only  did  they  begin 
to  enter  the  tubes.  When  a  strong  dose  of  cholera  vibrios  was 
injected  into  the  peritoneal  cavity  of  thoroughly  vaccinated  guinea- 
pigs,  the  animals  easily  resisted  the  inoculation.  When,  however, 
similarly  vaccinated  guinea-pigs  were  submitted  to  the  influence  of 
tincture  of  opium,  the  same  dose  of  vibrios  caused  their  death.  In 
these  narcotised  animals,  in  spite  of  the  considerable  dilatation  and 
hyperaemia  of  the  vessels  and  in  spite  of  the  marked  hyperleucocytosis 
of  the  blood,  diapedesis  was  not  produced  during  the  first  few  hours 
after  the  injection  of  theopium,  and  it  was  not  till  later  (5  to  6  hours 
after  injection)  that  the  leucocytes  began  to  appear  in  the  peritoneal 
cavity.  The  vibrios  profit  by  the  period  of  inactivity  of  the  phago- 
cytes to  multiply,  retaining  their  motility  and  also  the  property  of 
staining  with  basic  aniline  dyes.  When  the  retarded  leucocytes  make 
their  appearance  in  the  peritoneal  cavity,  they  find  it  already  invaded 
by  a  multitude  of  vibrios.  In  spite  of  this  the  leucocytes,  especially 
the  microphages,  ingest  an  enormous  number  of  the  organisms ;  this 
does  not  prevent  the  death  of  the  guinea-pigs,  though  it  takes  place 
some  hours  later  than  in  the  unvacciuated  control  animals.  At  the 
moment  of  death,  free  vibrios  are  no  longer  found  in  the  exu- 
dation ;  they  have  all  been  ingested  by  the  microphages,  inside 

1  Ann.  de  FInst.  Pasteur,  Paris,  1898,  t.  xn,  p.  288. 
B.  15 


226  Chapter  VIII 

which  they  have  undergone  granular  transformation.  On  making  a 
post-mortem  examination  of  the  animal  a  large  number  of  small 
heaps  of  vibrios,  such  as  are  never  met  with  in  animals  that  have 
not  been  submitted  to  the  action  of  opium,  are  found  on  the  omentum. 

All  that  is  necessary,  then,  is  to  retard  the  phagocytic  reaction 
for  a  few  hours  in  order  to  cause  well-vaccinated  guinea-pigs  to 
succumb  to  the  action  of  the  vibrios.  One  can  readily  understand 
[238]  that,  with  this  result  before  us,  there  can  be  no  hesitation  in  at- 
tributing to  phagocytosis  a  much  more  important  part  in  assuring 
acquired  immunity  than  to  Pfeiffer's  phenomenon. 

The  study  of  other  diseases  produced  by  vibrios  only  serves  to 
corroborate  the  general  conclusions  that  follow  from  the  detailed 
study  of  the  essential  processes  in  acquired  immunity  against  the 
cholera  vibrio.  It  is  here  necessary  to  recall  the  discovery  by 
v.  Behring  and  Nissen  of  the  very  marked  bactericidal  power  of 
the  blood  serum  of  guinea-pigs  that  have  been  vaccinated  against 
Oamaleia's  vibrio.  When  this  fact  was  first  demonstrated  we  were 
justified  in  thinking  that  the  vibrionicidai  property  of  the  blood 
might  by  itself  explain  this  acquired  immunity ;  but  a  comparative 
study  of  the  phenomena  which  take  place  in  vitro  with  those  which 
take  place  in  the  living  animal,  soon  demonstrated  how  slight  was 
the  foundation  for  this  hypothesis.  Whilst  the  vibrios,  when  sown 
in  the  blood  serum  of  hypervaccinated  guinea-pigs,  there  perished 
in  large  quantities  or  even  the  whole  of  them,  these  same  organisms, 
when  inoculated  into  the  subcutaneous  tissue  of  the  same  animals,  re- 
mained alive  for  several  days.  Gamaleia's  vibrio  is  much  less  capable 
of  being  transformed  into  granules  than  is  the  cholera  vibrio,  and  we 
find  it  retaining  its  normal  form  even  inside  the  leucocytes.  There  is 
no  occasion  in  this  case,  therefore,  to  look  for  Pfeiffer's  phenomenon. 

The  rapid  and  marked  destruction  of  Gamaleia's  vibrio,  in  vitro, 
in  the  blood  serum  of  vaccinated  guinea-pigs,  and  the  prolonged 
survival  of  these  organisms  in  the  living  animal,  afford  additional 
evidence  that  the  two  groups  of  phenomena  cannot  be  identical. 
On  the  other  hand,  it  furnishes  a  further  proof  that,  during  the 
preparation  of  the  serum,  there  is  produced,  parallel  with  the  co- 
agulation, another  process  which  confers  bactericidal  power  on  the 
serum.  It  is  quite  evident  that,  as  in  the  case  of  the  cholera  vibrio, 
we  have  here  to  do  with  the  liberation  of  microcytase  at  the 
expense  of  the  destroyed  or  injured  leucocytes.  Acting  along  with 
the  specific  fixative  of  the  body  fluids,  this  cytase  causes  the  death 


Facts  bearing  on  acquired  immunity  227 

of  the  vibrios  introduced  into  the  serum.  In  the  living  organism, 
the  microcytase  not  being  free,  these  vibrios,  although  influenced 
by  the  fixative,  resist  until  they  have  become  the  prey  of  the 
phagocytes.  In  an  investigation  which  was  the  subject  of  a  commu- 
nication to  the  International  Congress  of  Hygiene  in  London  in 
1891  \  I  demonstrated  that  the  phagocytic  reaction  is  produced  with  [239] 
great  intensity  in  guinea-pigs  that  have  been  vaccinated  against 
Gamaleia's  vibrio.  The  inoculation  of  this  organism  into  the  sub- 
cutaneous tissue,  an  inoculation  which  sets  up  a  rapidly  fatal  infection 
in  untreated  guinea-pigs,  gives  rise  in  immunised  animals  to  the 
formation  of  an  abundant  exudation,  in  which  the  numerous  vibrios 
soon  meet  with  resistance  from  the  phagocytes.  These  phagocytes 
ingest  the  living  vibrios,  retaining  them  for  some  considerable  time 
in  their  interior,  but  in  the  long  run  always  digesting  them  com- 
pletely. During  the  last  phase  of  this  struggle,  we  sometimes  find, 
inside  the  leucocytes,  vibrios  that  are  transformed  into  spherical 
granules.  It  was  with  these  cells,  filled  with  ingested  vibrios,  that 
I  was  able  first  to  carry  out  an  experiment  that  has  since  been 
repeated  again  and  again,  always  with  the  same  result.  When  from 
a  well- vaccinated  guinea-pig  a  drop  of  subcutaneous  exudation  is 
withdrawn,  at  a  stage  when  all  the  vibrios  have  for  some  time 
been  ingested  by  the  leucocytes,  and  transferred,  in  the  form  of 
a  hanging  drop,  to  the  incubator  at  35° — 37°  C.,  it  is  found  that  the 
ingested  vibrios  develop  inside  the  phagocytes  which  have  died  out- 
side the  animal.  The  vibrios  first  fill  the  leucocyte  and,  continuing 
to  multiply,  cause  the  cell  to  burst  when  they  distribute  themselves 
in  the  fluid  of  the  hanging  drop  (figs.  40  and  41).  This  experiment 
proves,  in  the  first  place,  that  the  vibrios  have  been  ingested  alive, 
and,  secondly,  that  the  plasma  of  the  exudation  was  incapable  of 
preventing  their  later  development. 

Having  summarised  the  principal  phenomena  exhibited  by  vibrios 
in  an  animal  possessing  acquired  immunity,  we  must  now  enquire 
whether  the  mode  of  destruction  and  disappearance  taking  place  in 
these  vibrios  is  of  general  application.  Naturally,  we  commence  this 
study  with  the  spirilla,  which  in  many  respects  present  a  great 
analogy  to  the  vibrios.  The  task  is  an  easy  one,  thanks  to  a  very 
careful  work  recently  published  by  Sawtchenko2,  on  the  Spirochaete 

1  [  Trans.  Seventh  Internat.  Congr.  of  Hyg.  and  Demogr.  London,  1892,  Vol.  n. 
p.  179 ;]  Ann.  de  llnst.  Pasteur,  Paris,  1891,  t.  v,  p.  465. 

2  Arch,  russes  de  Pathol.,  etc.,  St  Petersb.,  1900,  t.  ix,  p.  584;  Sawtchenko  et 
Melkich,  Ann.  de  llnst.  Pasteur,  Paris,  1901,  t.  xv,  p.  503. 

15—2 


228  Chapter   VIII 

obermeyeri  of  recurrent  fever.    We  know,  from  what  has  been  said  in 
Chapter  VI,  that  the  spirochaetes  found  in  the  serum  of  persons 


FIG.  40.    Vibrios  (V.  metchniTcovi)  developed  inside 
a  microphage  from  a  vaccinated  guinea-pig. 


FIG.  41.    Vibrios  (V.  metchniJcovi)  developed  in  a  drop  of  exudation 

from  a  vaccinated  guinea-pig.     The  vibrios  have  ruptured  the 

microphage  and  scattered  themselves  in  the  fluid. 

attacked  by  this  organism,  are,  in  the  peritoneal  cavity  of  guinea- 
pigs,  destroyed  by  the  intervention   of  the  macrophages.      These 
[240]  phagocytes  guarantee  the  natural  immunity  of  the  guinea-pig  against 


Facts  bearing  on  acquired  immunity  229 

the  parasite  of  recurrent  fever.  In  guinea-pigs,  into  which  blood 
or  serum  containing  spirilla  has  been  injected  on  several  occasions, 
the  destruction  of  these  micro-organisms  is  effected  in  a  different  [241] 
way.  When  Sawtchenko  introduced  a  number  of  Spirochaete  ober- 
meyeri  into  the  peritoneal  cavity  of  guinea-pigs  so  prepared,  he 
noted  that  they  underwent  a  transformation  resembling  that  ob- 
served in  Pfeiffer's  phenomenon.  In  a  short  time  the  majority  of 
these  micro-organisms  assumed  the  form  of  very  delicate  spirilla  to 
which  were  attached  round  granules.  There  was  not  a  complete 
transformation  of  the  spirilla  into  granules,  but  a  portion  of  their 
contents  exuded  in  the  form  of  spherical  drops.  The  spirilla  that 
exhibited  these  changes  lost  their  motility  and  collected  into  clumps. 
There  was  undoubtedly  an  extracellular  transformation  of  the  spirilla, 
but  this  took  place  only  in  the  peritoneal  cavity.  When  injected  into 
the  subcutaneous  tissue  of  a  prepared  guinea-pig  the  spirilla  brought 
about  the  formation  of  a  firm  but  scanty  exudation  in  this  situation. 
In  this  exudation  were  found  leucocytes  containing  spirochaetes  which 
retained  their  normal  form.  These  micro-organisms  were  found  ex- 
clusively in  macrophages  and  gave  no  evidence  of  the  occurrence 
of  Pfeiffer's  phenomenon.  A  like  absence  of  this  phenomenon  was 
observed  in  normal  guinea-pigs  which  had  been  injected  subcutane- 
ously  with  the  same  quantity  of  spirilla.  In  these  animals,  however, 
the  oedema  that  appeared  at  the  seat  of  inoculation  was  abundant 
and  soft,  and  the  disappearance  of  the  spirilla,  that  is  to  say  their 
ingestion  by  the  macrophages,  took  place  at  a  very  much  later  period 
than  in  the  prepared  guinea-pigs.  We  have,  therefore,  in  this  respect 
a  complete  analogy  with  the  vibrios :  in  both  cases  there  is  an  absence 
of  granular  transformation  below  the  skin  and  an  ingestion  by  the 
leucocytes  of  the  exudation;  on  the  other  hand,  we  have  Pfeiffer's 
phenomenon  appearing  in  the  peritoneal  fluid.  This  analogy  extends 
even  further.  Thus,  in  guinea-pigs  prepared  by  repeated  injections 
of  human  serum  rich  in  spirilla,  Sawtchenko  could  suppress  Pfeiffer's 
phenomenon  in  the  peritoneal  cavity  just  as  easily  as  in  the  case  of 
the  vibrios.  All  he  had  to  do  was  to  inject  a  certain  quantity 
of  broth  into  the  peritoneal  cavity  of  his  immunised  guinea-pigs. 
Twenty-four  hours  later,  on  introducing  spirilla  into  the  animals  at 
the  same  site,  they  retained  their  motility  for  hours,  did  not  exhibit 
any  granular  transformation  and  were  ultimately  completely  ingested 
by  the  macrophages. 

These  facts  lead  us  to  conclude  that  the  fate  of  the  spirochaetes 


230  Chapter  VIII 

[242]  of  recurrent  fever  in  the  organism  of  guinea-pigs  prepared  by  pre- 
vious injections  is  governed  by  laws  the  same  as  those  established 
for  acquired  immunity  against  vibrios.  The  spirilla  are  ingested  and 
destroyed  by  the  phagocytes,  except  where  phagolysis  occurs,  in 
which  case  the  cytase,  being  set  free,  attacks  the  micro-organisms 
outside  the  leucocytes. 

After  his  discovery  of  the  granular  transformation  of  vibrios, 
R.  Pfeiffer,  in  collaboration  with  several  of  his  pupils,  set  himself  to 
discover  how  far  this  phenomenon  was  general  in  acquired  immunity. 
He  directed  his  attention  to  the  typhoid  cocco-bacillus,  upon  which 
he  had  already  published1  a  very  detailed  account  of  work  carried 
out  in  conjunction  with  Kolle.  These  observers  availed  themselves 
of  the  discovery  made  by  Beumer  and  Peiper2,  and  Chantemesse  and 
Widal3  and  confirmed  by  other  observers,  that  laboratory  animals, 
especially  mice  and  guinea-pigs,  could  be  easily  vaccinated  against 
the  fatal  disease  set  u^  by  the  micro-organism  of  typhoid  fever.  As 
in  the  experimental  infection  of  the  guinea-pig  by  the  cholera  vibrio, 
the  vaccination  of  the  animals  against  the  typhoid  bacillus  could  be 
carried  out  very  easily,  either  by  using  sterilised  cultures  or  the  fluids 
of  cultures  deprived  of  their  organisms  by  filtration.  In  the  small 
laboratory  animals  a  most  marked  acquired  immunity  may  thus  be 
obtained,  and  the  study  of  the  phenomena  which  appear  in  the 
vaccinated  organism  afforded  evidence  of  a  general  analogy  with  those 
which  have  been  observed  when  vibrios  are  used.  In  the  peritoneal 
cavity  of  the  immunised  guinea-pigs,  Pfeifier's  phenomenon  proper 
does  not  appear,  that  is  to  say,  only  a  few  of  the  bacilli  are  trans- 
formed into  granules,  the  large  majority  retaining  their  bacillary 
form ;  still  they  are  evidently  greatly  damaged :  they  become  motion- 
less and  agglutinate  more  or  less  completely  into  clumps.  If,  however, 
a  few  of  these  micro-organisms  are  sown  on  nutritive  media,  they 
multiply  freely  and  give  abundant  growths.  The  peritoneal  fluid, 
then,  acts  most  unmistakably  upon  the  typhoid  bacillus,  but  in 
a  much  less  degree  than  does  the  peritoneal  exudation  of  guinea- 
pigs  upon  the  cholera  vibrio  when  immunised  against  that  organism. 

[243]  In  both  cases  we  have  a  pronounced  phagolysis  which  sets  free  the 
microcytase,  whose  action  on  the  vibrio  is  more  marked  than  on 
the  bacillus  of  typhoid  fever.  This  extracellular  action  on  the 

1  Ztschr.f.  Hyg.,  Leipzig,  1896,  Bd.  xxi,  S.  203. 

2  Ibid.  1887,  Bd.  n,  S.  110. 

3  Ann.  de  I'Inst.  Pasteur,  Paris,  1892,  t.  vi,  p.  755. 


Facts  bearing  on  acquired  immunity  231 

typhoid  bacillus  in  the  peritoneal  cavity  can  be  easily  prevented  by 
a  previous  injection,  twenty-four  hours  before,  of  broth,  physiological 
salt  solution,  or  normal  serum.  The  suppression  of  phagolysis  is,  as 
in  the  case  of  vibrios  and  spirilla,  followed  by  the  suppression  of 
extracellular  action  on  the  typhoid  bacilli. 

The  same  analogy  is  observed  in  the  phenomena  which  appear  be- 
neath the  skin.  The  bacillus  of  typhoid  fever,  when  introduced  into 
the  subcutaneous  tissue  of  vaccinated  guinea-pigs,  although  not  appre- 
ciably injured  by  the  fluid  of  the  exudation,  undergoes  some  agglu- 
tination. The  injurious  action  of  the  fluids  of  the  body  is  here  still 
less  effective  than  in  the  peritoneal  cavity.  But,  as  in  the  peritoneal 
cavity  of  vaccinated  guinea-pigs  previously  treated  with  broth,  so  in  the 
subcutaneous  exudation  it  is  the  phagocytes  which  destroy  the  micro- 
organisms. In  both  cases  there  is  a  very  great  afflux  of  leucocytes, 
mainly  microphages.  These  cells  ingest  and  digest  the  bacilli,  which 
ultimately  disappear.  The  micro-organisms  ingested  by  the  micro- 
phages, once  inside  these  phagocytes  are  transformed  into  granules 
very  like  those  observed  in  the  cholera  vibrio  similarly  treated.  In  this 
respect  the  analogy  between  the  two  micro-organisms  is  complete. 

Oppel,  working  in  my  laboratory,  has  repeated  Cantacuzene's 
work  on  the  retarding  action  of  opium  upon  the  phagocytic  process. 
He  obtained  the  same  results:  under  the  influence  of  the  narcotic, 
the  leucocytes  intervened  only  at  a  late  stage,  with  the  result  that 
the  vaccinated  guinea-pigs  succumbed  to  the  typhoid  infection.  The 
same  conclusion  must  be  drawn  from  the  experiments  made  by 
A.  Wassermann1.  Guinea-pigs  that  had  been  immunised  against  the 
bacillus  of  typhoid  fever  were  completely  resistant  to  a  dose  that  was 
always  fatal  to  the  control  animals.  When,  however,  along  with  this 
dose  of  bacilli,  a  certain  quantity  (3  c.c.)  of  a  serum  which  hinders 
the  phagocytic  reaction  is  injected,  the  guinea-pigs  lose  their  im- 
munity and  die  from  typhoid  infection.  The  serum  employed  by 
Wassermann  was  obtained  from  rabbits  that  had  been  treated  with 
the  blood  serum  of  guinea-pigs.  Rabbit's  serum,  thus  prepared, 
neutralises  the  action  of  the  guinea-pig's  cytase,  but,  as  demonstrated  [244] 
by  Besredka2,  it  also  exercises  several  other  functions,  one  especially, 
that  of  preventing  phagocytosis.  In  Wassermann's  experiments  it 
was  the  antiphagocytic  function,  then,  that  was  the  important  factor 

1  Ztschr.f.  Hyg.,  Leipzig,  1901,  Bd.  xxxvn,  S.  173. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  209. 


232  Chapter  VIII 

in  the  suppression  of  the  acquired  immunity  of  the  guinea-pigs. 
These  experiments  supply  a  fresh  proof  of  the  great  importance  of 
the  phagocytic  reaction  in  this  kind  of  immunity,  and  afford  further 
confirmation  of  the  analogy  between  the  mechanism  of  resistance  of 
the  animal's  organism  against  the  typhoid  bacillus  and  that  against 
the  cholera  vibrio. 

In  presence  of  this  striking  analogy,  it  is  unnecessary  to  insist 
further  on  the  details  of  the  acquired  immunity  of  animals  against 
the  experimental  disease  set  up  by  the  micro-organism  of  typhoid 
fever.  It  will  be  better  to  select  another  example  from  the  group 
of  bacilli.  Let  us  first  take  the  acquired  immunity  against  the 
bacillus  of  blue  pus  (Bacillus  pyocyaneus}  which  for  many  years  has 
been  regarded  as  the  best  example  in  which  to  study  this  kind  of 
immunity.  Charrin,  who  was  the  first  to  obtain  disease  with  this 
bacillus  experimentally,  published  several  notes1  on  the  acquired 
immunity  of  the  rabbit  against  it.  He  demonstrated  the  possibility 
of  vaccinating  this  animal  not  only  with  living  bacilli,  but  also  with 
the  products  of  their  culture;  he  studied  the  blood  serum  of  vacci- 
nated animals,  comparing  it  with  the  serum  of  normal  rabbits, 
especially  as  to  its  action  on  the  development  of  the  Bacillus  pyo- 
cyaneus. Although  unable  to  find  any  bactericidal  power  properly 
so  called  in  the  serum  of  immunised  rabbits,  Charrin  was  the  first 
to  draw  attention  to  certain  modifications  undergone  by  the  bacilli 
when  grown  in  this  medium.  He  noted  that  under  these  conditions 
110  pyocyanin  was  produced,  and,  in  collaboration  with  Roger,  he 
demonstrated  that,  in  the  serum  of  the  vaccinated  rabbit,  the  Bacillus 
pyocyaneus  forms  packets  composed  of  little  chains  of  greater  or 
less  length,  whilst  in  the  serum  of  the  normal,  susceptible  rabbit, 
it  develops  in  the  form  of  normal  rods,  the  rods  for  the  most  part 
being  isolated. 

From  his  experiments  in  vitro  Charrin  concluded  that  there  was 
marked  enfeeblement  of  the  functions  of  the  Bacillus  pyocyaneus 
when  submitted  to  the  action  of  the  vaccinated  animal  organism. 
[245]  Bouchard2  has  gone  so  far  as  to  develop  a  theory  of  acquired 
immunity,  in  which  the  principal  part  is  attributed  to  the  impos- 
sibility of  the  micro-organism,  after  it  has  invaded  the  refractory 
animal,  secreting  its  fluid  products;  there  is  no  vascular  dilatation 
and  diapedesis  does  not  take  place.  A  comparative  observation  of  the 

1  Compt.  rend.  Soc.  de  biol.,  Paris,  1889,  pp.  250,  330,  627  ;  1890,  pp.  203,  332, 195. 

2  "Les  microbes  pathogenes,"  Paris,  1892. 


Facts  bearing  on  acquired  immunity  283 

phenomena  observed  in  rabbits  that  are  susceptible  to  the  pyocyanic 
disease  and  of  those  met  with  in  vaccinated  rabbits,  most  clearly,  how- 
ever, demonstrates  the  impossibility  of  accepting  Bouchard's  interpre- 
tation. The  inoculation  of  the  bacillus  of  blue  pus  below  the  skin  of 
the  ear  of  the  normal  (un vaccinated)  rabbit  sets  up  extensive  inflam- 
matory reaction  with  marked  hyperaemia;  the  diapedesis  of  the  white 
corpuscles  takes  place  at  a  comparatively  late  stage  of  the  process 
and  phagocytosis  is  neither  set  up  nor  completed  until  very  late. 
On  the  other  hand,  in  vaccinated  rabbits,  infected  in  the  same  way, 
the  hyperaemia  of  the  ear  is  insignificant,  but  diapedesis  occurs  very 
early  and  phagocytosis  commences  at  once.  It  is  not,  therefore,  the 
impossibility  for  the  leucocytes  to  traverse  the  vessel  wall,  owing  to 
the  absence  of  the  dilatation  of  the  veins,  which  prevents  them  from 
making  their  way  rapidly  to  the  field  of  battle ;  it  is  their  imperfect 
positive  sensitiveness  that  is  accountable  for  the  tardy  and  incom- 
plete phagocytosis.  This  interpretation  is  confirmed  in  other  cases 
of  acquired  immunity. 

More  recently,  Paul  Muller1  has  laid  special  stress  on  the  part 
played  by  the  bactericidal  action  of  the  serum  of  animals  that  have 
been  vaccinated  against  the  pyocyanic  disease.  For  him  the  negative 
results  obtained  by  his  predecessors  lose  their  significance,  since  all 
their  experiments  were  carried  out  under  conditions  of  aerobiosis, 
whilst  it  is  only  in  the  absence  of  free  oxygen  that  this  bactericidal 
power  can  be  exerted  at  all  freely.  Muller,  therefore,  set  himself 
to  compare  under  anaerobic  conditions  the  bactericidal  action  on  the 
Bacillus  pyocyaneus  of  serums  coming  from  normal  and  from  vacci- 
nated animals.  He  succeeded  in  demonstrating  that  the  blood  serum 
of  vaccinated  animals  is  more  bactericidal  than  that  of  normal 
rabbits.  Before,  however,  drawing  any  conclusion  from  this  obser- 
vation, the  following  question  must  be  answered:  Are  the  phenomena 
observed  in  vitro  comparable  with  those  seen  in  the  living  animal? 
In  preceding  chapters  it  has  been  shown  so  often  that  the  blood 
serum  obtained  after  the  separation  of  the  extravascular  clot,  can 
in  no  way  be  identified  with  the  plasma  of  the  circulating  blood,  [246] 
that  it  is  unnecessary  to  argue  this  matter  further.  If  we  wish 
to  gain  a  clear  idea  of  the  mechanism  of  immunity  in  the  living 
animal  we  must  observe  the  course  of  events  in  the  vaccinated 
animal  and  not  draw  conclusions  from  observations  in  vitro  except 
after  strict  examination.  All  the  works  on  pyocyanic  immunity 

1  Cenlralbl.  f.  Bakteriol  u.  Parasitenk.,  lte  Abt.,  Jena,  1900,  Bd.  xxvm,  S.  577. 


234  Chapter  VIII 

above  summarised  lie  under  the  reproach  that  in  them  this  rule 
has  not  been  adhered  to. 

Since  the  discovery  of  Pfeiffer's  phenomenon  in  animals  that  have 
been  vaccinated  against  the  cholera  vibrio,  much  greater  care  has 
been  taken  to  attend  to  the  changes  that  occur  in  the  animal  that 
enjoys  acquired  immunity.  Wassermann1  was  the  first  to  attempt 
to  apply  Pfeiffer's  discovery  to  the  Bacillus  pyocyanem.  With  a  race 
of  this  bacillus  rendered  more  virulent  he  succeeded  in  producing  a 
fatal  experimental  malady  in  the  guinea-pig  against  which  he  was 
able  by  various  methods  to  vaccinate  these  animals. 

He  thus  describes  the  phenomena  observed  in  the  peritoneal 
cavity  of  immunised  guinea-pigs.  Soon  after  injection  the  bacilli 
of  blue  pus  become  motionless,  then  "the  rods  swell  up  and  melt, 
like  wax  in  hot  water.  The  formation  of  granules,  such  as  occur  in 
the  cholera  vibrio,  has  been  observed  but  rarely.  The  process  recalls 
rather  that  which  takes  place  in  experimental  typhoid  fever,  as 
described  by  R.  Pfeiffer.  In  all  cases  the  phenomenon  of  solution  takes 
place  entirely  in  the  fluid  of  the  exudation,  without  any  co-opera- 
tion on  the  part  of  the  leucocytes"  (p.  284).  We  see  that  we  have 
still  to  do  with  a  kind  of  attenuated  Pfeiffer's  phenomenon,  without 
any  granular  change,  but  with  an  immobilisation  of  the  bacilli.  As 
Wassermann  has  remained  satisfied  with  the  examination  of  the 
peritoneal  content  which,  as  we  know,  gives  but  an  imperfect  picture 
,  of  acquired  immunity,  Gheorghiewsky2  set  himself  to  study  the 
question  more  thoroughly  under  my  direction.  With  this  object  he 
vaccinated  a  series  of  guinea-pigs  with  living  bacilli  of  blue  pus, 
a  sure  method  of  obtaining  acquired  immunity.  On  examining  the 
peritoneal  fluid  (withdrawn  shortly  after  the  injection  of  the  bacilli) 
of  the  vaccinated  guinea-pigs,  he  found  that  the  bacilli  were  motion- 
less and  had  undergone  a  certain  degree  of  agglutination.  They  were 
[247]  not  transformed  into  granules  but  became  thicker  and  somewhat  more 
dumpy.  These  changes  are  observed  during  the  period  of  phago- 
lysis,  when  only  a  few  scattered  leucocytes  are  to  be  found  in  the 
fluid  of  the  peritoneal  cavity.  About  two  hours  after  the  injection 
of  the  bacilli  the  leucocytes  begin  to  reappear  in  the  peritoneal 
exudation,  more  especially  the  microphages,  which  lose  no  time  in 
seizing  the  bacilli,  some  of  which  become  transformed  into  granules. 
A  few  hours  later  the  exudation,  containing  a  multitude  of  leucocytes, 

1  Ztschr.f.  Hyg.,  Leipzig,  1896,  Bd.  xxii,  S.  263. 

2  Ann.  de  Flnst.  Pasteur,  Paris,  1899,  t.  xm,  p.  298. 


Facts  tearing  on  acquired  immunity  235 

no  longer  contains  any  free  bacilli:  all  are  found  inside  the  micro- 
phages.  Nevertheless,  if  a  drop  of  the  exudation  now  be  withdrawn 
and  kept  for  some  time  at  a  temperature  of  37°  C.,  it  will  be  found 
that  the  bacilli  multiply  inside  the  dead  phagocytes  outside  the 
animal.  We  thus  obtain  colonies  of  bacilli,  a  fact  which  clearly 
proves  that  these  bacilli  whilst  still  alive  have  been  ingested  by  the 
leucocytes.  This  experiment  is,  therefore,  very  similar  to  the  one 
we  have  described  in  connection  with  Gamaleia's  vibrio. 

Even  at  a  later  period,  24  or  30  hours  after  the  injection  of  the 
bacilli,  that  is  to  say  at  a  period  when  an  examination  of  the  exu- 
dation no  longer  reveals  the  presence  of  bacilli,  the  sowing  of  a  drop 
of  this  exudation  on  a  nutrient  medium  still  gives  isolated  colonies  of 
the  Bacillus  pyocyaneus  capable  of  producing  the  characteristic 
pigments.  At  a  still  later  period,  when  the  peritoneal  exudation 
remains  sterile,  a  post-mortem  examination  of  the  animals  enables 
one  to  recognise,  beneath  the  peritoneal  surface,  small  white  points 
made  up  of  leucocytes.  The  sowing  of  these  masses  almost  invariably 
gives  colonies  of  the  Bacillus  pyocyaneus  which  form  blue  pigments. 
We  see  from  this  account  that,  even  in  the  peritoneal  cavity  of 
vaccinated  animals,  matters  by  no  means  go  on  in  a  uniform  fashion, 
as  would  appear  from  Wassermann's  statements.  Some  bactericidal 
action  in  the  peritoneal  fluid  there  certainly  is,  but  it  is  quite 
transient,  and  is  limited  to  the  period  of  phagolysis.  The  majority 
of  the  bacilli  resist  this  attack  of  the  body  fluids  to  continue  their 
struggle  with  the  phagocytes,  which,  however,  ultimately  get  the 
upper  hand.  In  the  subcutaneous  tissue  the  part  played  by  this 
phagocytic  reaction  is  still  more  general.  Gheorghiewsky  has  studied 
it  not  only  in  vaccinated  guinea-pigs  but  also  in  a  goat  which  had 
received  several  large  injections  of  the  Bacillus  pyocyaneus.  He 
observed  that  shortly  after  the  subcutaneous  injection  of  these  bacilli, 
the  fluid  which  accumulates  at  the  seat  of  inoculation  renders  them  [248] 
motionless  and  in  part  agglutinates  them.  This  fluid  is  clear  and 
contains  a  few  leucocytes  and  a  number  of  bacilli  which  still  retain 
their  usual  form.  Some  time  later  the  leucocytes  begin  to  come 
up  to  the  seat  of  inoculation  and  to  ingest  the  bacilli.  At  the  end 
of  10  to  15  hours  all  the  bacteria  have  been  seized  by  the  micro- 
phages  and  we  no  longer  find  any  of  them  free.  A  hanging  drop 
of  this  exudation,  transported  to  the  incubator,  soon  swarms  with 
bacilli  which  have  sprung  from  the  organisms  ingested  by  the  leu- 
cocytes. 


236  Chapter  VIII 

The  exudation  becomes  more  and  more  abundant  at  the  seat  of 
inoculation  and  ends  in  the  formation  of  an  abscess,  from  the  contents 
of  which  cultures  of  the  Bacillus  pyocyaneus  may  be  obtained  for 
a  fortnight.  The  bacilli,  however,  finally  disappear,  this  being  due 
to  the  destructive  action  of  the  phagocytes  and  not  to  that  of  the 
fluid  of  the  exudation. 

This  fundamental  part  played  by  phagocytosis  in  acquired  im- 
munity against  the  Bacillus  pyocyaneus  has  been  confirmed  by 
Gheorghiewsky  by  experiments  on  guinea-pigs  vaccinated  and  then 
submitted  to  the  action  of  opium.  As  in  the  analogous  experiments 
of  Cantacuzene  on  the  cholera  vibrio,  the  opium  narcosis  retards  dia- 
pedesis  and  this,  for  some  time,  increases  the  chances  of  the  bacilli. 
A  tardy  diapedesis  and  phagocytosis,  no  doubt,  is  produced  which  ends 
in  the  ingestion  of  the  bacilli,  but  the  animal  loses  its  acquired 
immunity  and  finally  succumbs  in  spite  of  the  fact  that  the  dose  of 
Bacillus  pyocyaneus  was  insufficient  to  kill  a  control  guinea-pig  vac- 
cinated to  the  same  degree,  but  not  submitted  to  the  action  of  opium. 

The  example  we  have  just  analysed  relates,  then,  to  a  micro- 
organism which  is  more  resistant  than  are  the  vibrios,  Obermeyer's 
spirilla  or  even  the  typhoid  bacillus,  to  the  action  of  the  microcytase 
which  has  escaped  from  the  cells  during  phagolysis.  The  Bacillus 
pyocyaneus  undergoes,  in  the  fluids  of  the  vaccinated  animal,  the 
action  of  the  specific  fixative  and  can  thus  be  rendered  motionless 
and  become  agglutinated.  But  this  action  is  insufficient  to  ensure 
immunity  and  should  phagocytosis  not  take  place  in  time  to  ingest 
the  bacilli,  the  vaccinated  animal  succumbs.  The  reaction  of  the 
phagocytes  is,  therefore,  indispensable  if  the  acquired  immunity  is 
to  be  effective.  In  this  respect  the  analogy  is  very  great  between 
the  resistance  of  the  vaccinated  animal  against  the  various  bacteria 
(vibrios,  spirochaetes,  typhoid  cocco-bacilli,  bacilli  of  blue  pus)  that 
we  have  so  far  studied  in  this  chapter.  These  bacteria  have,  however, 
[249] this  in  common; — they  are  all  endowed  with  a  considerable  power 
of  motion.  Pursuing  our  examination  of  the  principal  data  on 
acquired  immunity  against  micro-organisms,  we  must  now  choose 
examples  from  the  group  of  non-motile  bacilli;  amongst  these  we 
assign  the  first  place  to  the  micro-organism  of  swine  erysipelas.  This 
small  bacillus  has  been  the  subject  of  several  important  researches 
on  acquired  immunity,  one  of  which  at  a  certain  period  caused  quite 
a  sensation  in  the  bacteriological  world.  Emmerich1,  in  an  investi- 
1  Fortschr.  d.  Med.,  Berlin,  1888,  Bd.  vi,  S.  729. 


Facts  bearing  on  acquired  immunity  237 

gation  carried  out  in  collaboration  with  di  Mattel,  made  an  unexpected 
announcement.  He  said  he  believed  that  he  was  justified  in  affirm- 
ing that  the  acquired  immunity  of  rabbits  against  the  bacillus  of 
swine  erysipelas  is  due  to  the  formation,  in  the  fluids  of  the  body,  of 
an  antiseptic  substance  which  very  quickly  destroys  this  organism. 
This  substance,  secreted  by  the  cells  of  the  vaccinated  animal,  was 
supposed  to  act  after  the  fashion  of  a  solution  of  bichloride  of  mercury 
and  to  kill  a  large  number  of  bacilli,  introduced  subcutaneously,  in  from 
15  to  25  minutes.  This  discovery  was  not  confirmed.  In  a  series 
of  experiments  that  I  carried  out1  with  the  object  of  clearing  up 
this  question,  and  made  under  conditions  as  favourable  as  possible 
for  the  demonstration  of  the  supposed  bactericidal  secretion,  this 
action  was  never  manifested.  Not  only  did  the  virulent  bacilli  of 
swine  erysipelas,  when  injected  subcutaneously  into  well  vaccinated 
rabbits,  remain  alive  in  the  subcutaneous  exudation  for  hours  and 
even  days,  but  the  attenuated  bacilli  of  Pasteur's  vaccines  likewise 
remained  intact.  These  bacilli  when  introduced  into  the  anterior 
chamber  of  the  eye  survived  for  even  a  longer  period.  Here,  as 
beneath  the  skin,  the  injection  of  the  bacilli  induced  an  exudation 
rich  in  leucocytes,  amongst  which  microphages  predominated.  These 
phagocytes  at  once  began  to  seize  the  bacilli  which  were  destroyed 
not  in  the  fluid  of  the  exudation  but  within  the  leucocytes.  Long 
after  all  the  bacilli  had  been  ingested,  24  hours  and  more  after 
inoculation,  the  sowing  of  the  exudation  frequently  gave  growths 
in  appropriate  media. 

Emmerich2  sought  by  new  experiments  to  remove  these  objections, 
but  he  found  that  the  bacilli  of  swine  erysipelas  did  not  disappear  [250] 
from  the  vaccinated  animal  until  some  8  or  10  hours  after  they 
had  been  introduced.  There  is,  therefore,  no  longer  any  question 
of  a  rapid  bactericidal  action  at  all  comparable  to  that  of  corrosive 
sublimate,  which  would  destroy  the  introduced  bacilli  in  less  than  an 
hour.  The  limit  of  8  to  10  hours,  accepted  by  Emmerich,  is  still  too 
short  and  is  not  in  accordance  with  my  experiments ;  but  even  this  was 
quite  sufficient  for  the  appearance  of  a  free  phagocytosis,  a  condition 
that  really  occurs.  Emmerich  has  not  directed  his  researches  in  this 
direction,  and  his  theoretical  conclusions  did  not  in  the  least  weaken 
the  value  of  my  arguments  drawn  from  the  demonstration  of  the 
ingestion  and  intracellular  destruction  of  the  bacilli  by  phagocytes. 

1  Ann.  de  VInst.  Pasteur^  Paris,  1889,  t.  ni,  p.  289. 

2  Arck.f.  Hyg.,  Miinchen  u.  Leipzig,  1891,  Bd.  xn,  S.  275. 


238  Chapter  VIII 

The  researches  on  immunity  against  swine  erysipelas  then  lan- 
guished for  some  time,  until  the  discovery  of  Pfeiffer's  phenomenon 
gave  a  fresh  stimulus  to  the  study  of  this  problem.  One  of  Pfeiffer's 
pupils,  Voges1,  sought  to  apply  the  results  obtained  in  the  case  of 
the  cholera  vibrio  to  the  acquired  immunity  against  the  bacillus 
of  swine  erysipelas.  He  studied  the  blood  serum  of  animals  vacci- 
nated against  this  bacillus  and  believed  himself  justified  in  affirming 
the  existence  of  an  acquired  bactericidal  power.  Under  no  con- 
dition, however,  did  he  observe  anything  comparable  to  Pfeiffer's 
phenomenon,  and  he  was  compelled  to  admit  that  the  bactericidal 
action  of  the  serum  is  very  feeble  and  only  takes  effect  on  young 
bacilli  whose  membranes  are  as  yet  very  delicate  and  not  very 
resistant.  Mesnil2  repeated  these  researches  in  my  laboratory,  but 
his  results  were  very  different  from  those  obtained  by  Voges.  The 
blood  serum  of  rabbits,  fully  vaccinated  against  the  bacillus  of  swine 
erysipelas,  proved  to  be  a  good  culture  medium  for  this  bacillus,  and 
Mesnil  affirms,  as  the  result  of  numerous  well-established  obser- 
vations, that  "in  vitro,  the  serum  of  rabbits  immunised  against  the 
erysipelas  has  no  bactericidal  power  or  a  very  insignificant  one."  On 
the  other  hand,  the  same  fluid  had  a  very  marked  agglutinative 
power.  The  bacillus  of  swine  erysipelas,  being  non-motile,  does  not 
present  the  abrupt  change  that  is  observed  in  vibrios  or  in  the 
typhoid  bacillus  when  submitted  to  the  influence  of  specific  serums— 
under  which  conditions  these  organisms  at  once  lose  their  motility. 
But  the  bacilli  of  swine  erysipelas,  when  introduced  into  the  specific 
serum  of  vaccinated  animals,  run  together  into  masses  which  become 
[251]  more  and  more  voluminous  and  fall  to  the  bottom  of  the  vessel, 
leaving  a  limpid  supernatant  fluid.  When  this  bacillus  is  sown  in 
the  serum  of  vaccinated  animals,  it  is  seen  to  develop  in  the  form 
of  chains,  composed  of  a  large  number  of  segments,  which  fall  to 
the  bottom  of  the  tube.  These  bacilli,  however,  whether  agglutinated 
or  developed  in  chains,  never  show  any  attenuation  in  virulence. 
When  the  serum  which  bathes  them  is  got  rid  of  by  washing,  they 
are  just  as  virulent  as  are  the  bacilli  developed  in  the  serum  of 
normal  unvaccinated  rabbits.  It  is  important  to  show  that  this 
virulence  is  kept  up  in  spite  of  the  fact  that  the  bacilli,  when  placed 
in  contact  with  the  serum  of  immunised  animals,  become  permeated 

1  Ztschr.f.  Hyg.,  Leipzig,  1896,  Bd.  xxn,  S.  515;  Deutsche  med.   Wchnschr., 
Leipzig,  1898,  S.  49 ;  Ztschr.f.  Hyg.,  Leipzig,  1898,  Bd.  xxvm,  S.  38. 

2  Ann.  de  I'Inst.  Pasteur,  Paris,  1898,  t.  xu,  p.  481. 


Facts  bearing  on  acquired  immunity  239 

with  the  specific  fixative,  as  shown  by  the  experiments  of  Bordet 
and  Gengou1.  These  observers,  indeed,  have  demonstrated  that  the 
bacilli  of  swine  erysipelas,  when  kept  for  24  hours  in  the  specific 
serum  heated  to  55°  C.,  acquire  the  property  of  absorbing  the  cytases 
contained  in  the  unheated  serum  of  normal  animals. 

The  study  of  acquired  immunity  against  the  bacillus  of  swine 
erysipelas  teaches  us  that  this  immunity  is  not  due  to  any  extra- 
cellular destruction  comparable  with  Pfeiffer's  phenomenon ;  and  that 
this  immunity  causes  the  production  of  a  specific  fixative  and  of  a 
specific  agglutinative  substance,  whose  action  on  the  resistance  of  the 
animal,  to  judge  from  the  complete  virulence  of  the  bacilli  when 
agglutinated  and  impregnated  by  fixative,  is  feeble  or  nil.  It  is 
the  phagocytic  reaction  which  is  dominant  in  the  immunised  animals 
and  which  brings  about  the  intracellular  destruction  of  the  bacilli. 

The  history  of  the  anthrax  bacillus,  another  representative  of  the 
group  of  non-motile  bacilli,  is  particularly  interesting,  the  more  so 
that  for  some  time  the  researches  on  acquired  immunity  have  been 
concentrated  almost  entirely  on  the  analysis  of  the  facts  observed 
in  animals  that  have  been  vaccinated  with  the  two  Pasteur  vaccines. 
In  this  way  a  large  number  of  valuable  facts  have  been  collected; 
of  these  the  more  important  may  be  presented  to  the  reader. 

In  my  first  work  on  this  subject2  I  called  attention  to  the  fact 
that  in  the  rabbit  vaccinated  against  anthrax,  the  bacilli,  when 
inoculated  subcutaneously,  soon  become  the  prey  of  leucocytes  which 
accumulate  at  the  spot  menaced.  In  the  unvaccinated  control 
rabbits,  however,  the  anthrax  bacilli  remain  in  a  free  state  in  the 
fluid  of  the  subcutaneous  exudation,  only  a  few  isolated  rods  being  [252] 
found  inside  phagocytes.  I  have  since  been  able  to  confirm  this 
fact8,  which  must  now  be  regarded  as  fully  established.  In  the 
vaccinated  rabbits  the  leucocytes  exhibit  a  very  marked  positive 
chemiotaxis  against  the  anthrax  bacilli,  whilst  in  normal  unvacci- 
nated rabbits  the  chemiotaxis  of  the  leucocytes  in  the  anthrax  of 
the  subcutaneous  tissue  is  distinctly  negative.  When  a  small  quantity 
of  anthrax  culture  is  inoculated  subcutaneously  into  vaccinated  and 
into  unvaccinated  rabbits  there  may  be  observed,  even  within  a  few 
hours,  a  very  great  difference.  In  the  former  there  is  found  at  the 
seat  of  inoculation  an  infiltration  which  swarms  with  leucocytes 

1  Ann.  de  Flnst.  Pasteur,  Paris,  1901,  t.  xv,  p.  295. 

2  Vir chow's  Archiv,  Berlin,  1884,  Bd.  xcvii,  S.  502. 

3  Virchow's  Arc/iw,  Berlin,  1888,  Bd.  cxiv,  S.  465. 


240  Chapter  VIII 

in  the  act  of  devouring  bacilli.  In  the  normal,  susceptible  rabbit, 
on  the  other  hand,  the  exudation  produced  is  soft,  rich  in  fluid,  and 
very  poor  in  leucocytes.  The  vessels  in  the  vicinity  are  distended  with 
blood,  and  the  fact  that  the  leucocytes  do  not  come  up  to  the  seat  of 
inoculation  is  in  no  way  due  to  the  absence  of  vascular  dilatation 
which  might  prevent  diapedesis.  The  vessels  are  much  more  dilated 
than  in  the  vaccinated  rabbit,  and  yet  in  the  latter  the  emigration  is 
incomparably  greater.  This  essential  difference  must  be  attributed 
to  the  sensitiveness  of  the  leucocytes,  which  exhibit  a  negative 
chemiotaxis  in  the  normal  rabbit  but  a  very  marked  positive  chemio- 
taxis  in  the  vaccinated  rabbit. 

It  has  been  shown  repeatedly  that  the  subcutaneous  exudation, 
very  rich  in  leucocytes  which  have  had  time  to  ingest  all  the  bacilli, 
when  inoculated  into  guinea-pigs,  ensures  the  appearance  in  them 
of  a  generalised  and  fatal  anthrax;  this  affords  evidence  that  the 
phagocytosis  is  exercised  against  virulent  and  therefore  living  bacilli. 
Marchoux1,  in  Roux's  laboratory,  has  carried  out  numerous  experi- 
ments on  the  vaccination  of  rabbits  and  has  observed  that  the 
inoculated  anthrax  bacilli  cause  an  exudation  very  rich  in  leucocytes, 
and  that  these  cells  ingest  and  destroy  the  bacilli.  The  phagocytes 
easily  rid  the  refractory  animal  of  the  bacilli  in  the  vegetative  state, 
but  the  spores  are  much  more  resistant.  After  being  devoured  by  the 
leucocytes  they  may  remain  inside  them  for  months  without  germinat- 
ing. Marchoux  obtained  cultures  of  anthrax  from  the  subcutaneous 
exudation  taken  from  vaccinated  rabbits  70  days  after  inoculation. 

The  fact  that  the  bactericidal  action  of  the  blood  serum  on 
[253]  anthrax  bacilli  is  specially  well  marked  in  the  rat,  suggested  the 
idea  of  trying  to  obtain,  in  this  rodent,  an  augmentation  of  this 
property  as  a  result  of  vaccination.  Sawtchenko2  attempted  to  do 
this  in  an  investigation  already  cited  in  Chapter  VI,  carried  out  in 
my  laboratory.  He  succeeded  in  thoroughly  vaccinating  white  rats 
against  virulent  anthrax  and  in  showing  that  the  blood  serum  of  these 
animals  rendered  refractory  "  is  bactericidal  in  the  same  degree  as  that 
of  non-immunised  rats."  In  the  vaccinated  rats  "the  subcutaneous 
exudation  was  as  free  from  bactericidal  substances  as  was  the  lymph 
of  the  control  animals."  Sawtchenko  was  unable  to  demonstrate  any 
increase  of  bactericidal  power  except  in  the  peritoneal  exudation  of 
rats  vaccinated  by  injection  of  cultures  into  the  peritoneal  cavity. 

1  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  805. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1897,  t.  xi,  p.  881. 


Facts  bearing  on  acquired  immunity  241 

In  spite,  however,  of  the  absence  of  any  increase  in  the  bacteri- 
cidal property  of  the  blood  serum  and  of  the  subcutaneous  exudation 
in  vaccinated  rats,  the  cell  reaction  obtained  in  them  is  very 
different  from  that  met  with  in  normal,  susceptible  rats.  In  a  very 
short  time  (3  to  5  hours)  after  the  subcutaneous  injection  of  anthrax 
bacilli  into  the  control  rats  (susceptible),  an  evident  oedema  is 
produced;  in  the  vaccinated  rat  there  is  none.  The  exudation, 
not  very  abundant  in  the  latter,  already  contains  a  number  of  leuco- 
cytes which  are  actively  phagocytic,  whilst  in  the  control  animal, 
examined  simultaneously,  "leucocytes  are  rarely  met  with,  and  few 
of  them  contain  bacilli."  Later,  the  difference  becomes  still  more 
marked.  Pronounced  oedema  occurs  in  the  control  animal,  it  is  poor 
in  leucocytes  but  rich  in  bacilli,  which  continue  to  multiply;  but 
"  in  the  immunised  rat,  we  find  not  a  clear  exudation  but  a  thick  and 
purulent  fluid,  full  of  leucocytes."  These  cells  devour  all  the  bacilli ; 
not  a  single  one  remains  free.  "Even  after  14  hours  bacilli  ingested 
by  the  leucocytes  are  present  and  a  culture  of  anthrax  bacilli  may  be 
obtained  from  fluid  taken  from  the  seat  of  inoculation.  Further, 
guinea-pigs  or  rats,  when  inoculated  with  a  drop  of  this  exudation 
(which  contains  no  anthrax  spores),  succumb  to  anthrax." 

Even  before  these  researches  on  the  immunity  of  rats  had  been 
carried  out,  an  attempt  had  been  made  to  gain  some  idea  of  the 
differences  presented  by  the  vaccinated  fluids  of  animals  as  compared 
with  those  presented  by  the  fluids  of  control  animals  susceptible  to 
anthrax.  In  1886  I  was  able  to  demonstrate1  that  the  anthrax  [25<t] 
bacillus  develops  abundantly  in  the  defibrinated  blood  of  sheep  that 
had  acquired  immunity  as  the  result  of  vaccination  by  Pasteur's 
method.  When  these  bacilli  contain  spores  and  are  inoculated  into 
rabbits  they  rapidly  produce  a  fatal  anthrax;  but  when  no  spores 
are  present  the  injection  of  bacilli  does  not  produce  a  fatal  disease, 
and  such  infection  is  well  supported  by  the  rabbits.  From  this  I 
concluded  at  that  time  that  the  anthrax  bacillus  must,  in  the  blood 
of  the  vaccinated  sheep,  undergo  a  real  attenuation  in  virulence,  an 
interpretation  which,  as  will  be  seen  in  the  next  chapter,  was  found 
to  be  erroneous. 

Nuttall2  showed  that  the  defibrinated  blood  of  refractory  sheep 
acted  as  a  nutrient  medium  for  the  anthrax  bacillus.  Making  com- 
parative investigations,  by  the  plate  method,  on  the  bactericidal 

1  Ann.  de  FInst.  Pasteur,  Paris,  1887, 1. 1,  p.  42. 

2  Ztschr.f.  Hyg.,  Leipzig,  1888,  Bd.  iv,  S.  353. 

B.  16 


242  Chapter  VIII 

power  of  the  blood  of  vaccinated  and  normal  sheep,  he  observed  that, 
in  both  cases,  there  was,  at  first,  a  certain  decrease  in  the  number  of 
bacilli  sown,  more  marked  in  the  blood  of  the  vaccinated  than  in 
that  of  the  control  animals.  Nevertheless,  8  hours  after  the  com- 
mencement of  the  experiment  the  anthrax  bacteria  had  produced 
innumerable  bacilli  in  the  blood  of  the  refractory  sheep.  Nuttall 
satisfied  himself  that  this  feeble  bactericidal  power  was  not  to  be 
compared  with  the  very  much  greater  power  of  the  blood  of  the 
rabbit,  an  animal  specially  susceptible  to  anthrax. 

More  recently  the  properties  of  the  serum  of  sheep  which  have 
been  vaccinated  against  anthrax  have  been  studied  very  carefully  by 
Sobernheim1.  He  also  was  able  to  show  that  this  serum  allows  of  an 
abundant  development  of  the  bacillus,  and  that,  outside  the  animal, 
it  does  not  exercise  any  more  appreciable  bactericidal  power  than 
does  the  serum  of  the  normal  sheep.  The  serum  of  the  best  vacci- 
nated sheep  was  found  to  be  incapable  of  destroying  even  very  small 
quantities  of  anthrax  bacilli.  The  only  change  that  Sobernheim  could 
make  out  was  with  regard  to  the  thickening  of  the  bacterial 
membrane.  This  modification,  however,  was  not  constant  and  could 
not  be  seen  in  the  serum  of  certain  vaccinated  sheep. 

The  serum  of  the  sheep  vaccinated  by  Sobernheim  exhibited  no 
[255]  increase  of  agglutinative  power  as  regards  virulent  bacilli.  Gengou2, 
however,  made  it  clear  that  repeated  injections  of  cultures  of  the 
first  vaccine  of  Pasteur  into  dogs  produced  a  marked  augmentation 
of  this  agglutinative  power ;  but  it  was  only  produced  when  the 
attenuated  bacillus  was  used.  The  virulent  anthrax  bacillus,  de- 
veloped as  isolated  rods,  was  not  affected  in  the  least  by  serum 
that  was  highly  agglutinative  for  the  bacillus  of  the  first  vaccine. 
Gengou  also  made  the  converse  experiment  with  the  serum  of  a 
dog  into  which  he  had  previously  injected  a  number  of  virulent 
anthrax  bacilli.  The  dog,  naturally  refractory  to  anthrax,  resisted 
the  inoculation  perfectly,  but  its  serum  did  not  acquire  any 
agglutinative  power  against  the  first  vaccine.  He  concluded 
therefrom  that  "the  part  played  by  agglutinins  in  the  defence  of 
the  animal  must  be  regarded  as  extremely  problematical"  (p.  339). 
On  the  other  hand  the  phagocytic  reaction  in  the  vaccinated  sheep 
is  always  very  pronounced  and  constant.  Von  Behring3,  in  one  of  his 

1  Ztschr.f.  Hyg.,  Leipzig,  1899,  Bd.  xxxi,  S.  89. 

2  Arch,  internal  de  Pharmacodyn.,  Gand  et  Paris,  1899,  Vol.  vi,  pp.  303,  338. 

3  "  Infectionsschutz  und  Iminunitat"  in  Eulenburg's  "  Real-Encyclopadie  d.  ges. 
Heilkunde,"  mte  Aufl.  (Encyclop.  Jahrbucher\  Wien,  1900,  Bd.  ix,  S.  202. 


Facts  bearing  on  acquired  immunity  243 

most  recent  publications,  expresses  the  opinion  that  this  example 
of  acquired  immunity  must  be  placed  in  the  category  of  phagocytic 
immunity. 

In  the  group  of  bacilli,  several  examples  of  which  we  have 
studied,  the  typhoid  bacillus  approaches  still  more  closely  to  the 
vibrios  and  spirilla  in  its  relation  to  humoral  properties.  Here 
may  be  observed  a  kind  of  attenuated  Pfeiffer's  phenomenon  and 
somewhat  profound  modifications  taking  place  under  the  influence 
of  the  serum  of  vaccinated  animals.  The  Bacillus  pyocymieus  is 
more  resistant  to  the  injurious  influence  of  fluids  taken  from  im- 
munised animals.  This  resistance  is  still  more  marked  in  the  bacillus 
of  swine  erysipelas  and  again  still  greater  in  the  anthrax  bacillus. 
Whilst,  however,  these  properties  of  the  fluids  of  the  body  are  found 
to  be  very  variable  and  of  unequal  power,  the  phagocytic  reaction 
is  constantly  manifested  and  always  very  actively.  The  leucocytes 
which,  in  susceptible  animals,  exhibit  a  very  marked  negative  chemio- 
taxis  or  only  a  tardy  and  incomplete  positive  chemiotaxis,  have, 
in  the  vaccinated  animal,  this  positive  susceptibility  developed  in 
a  very  high  degree. 

Before  quitting  the  group  of  bacteria  we  must  cast  a  glance  at 
the  mechanism  of  acquired  immunity  against  representatives  of  the  [256] 
group  of  spherical  micro-organisms.  Amongst  the  cocci  the  strepto- 
cocci have  been  especially  studied  as  regards  this  immunity.  For 
long  great  difficulties  were  encountered  in  vaccinating  animals 
against  these  chain  cocci,  but  Roger1,  Marmorek2,  Denys  and  Leclef3 
overcame  these  obstacles  and  succeeded  in  immunising  the  rabbit, 
one  of  the  most  susceptible  species,  to  their  pathogenic  action. 
More  recently  the  larger  mammals,  notably  the  horse,  have  been 
successfully  immunised.  A  certain  number  of  important  facts,  the 
knowledge  of  "which  is  useful  to  complete  the  survey  of  the  pheno- 
mena of  acquired  immunity,  have  thus  been  collected. 

Roger  set  himself  to  study  the  properties  of  the  blood  serum 
of  rabbits  vaccinated  against  the  streptococcus,  and  established 
the  fact  that  this  fluid  had  not  the  slightest  appreciable  bacteri- 
cidal action ;  the  streptococcus  grew  in  it  just  as  well  as  in  the  serum 

1  Compt.  rend.  Sec.  de  biol.,  Paris,  1891,  p.  538 ;  1895,  pp.  124,  224;  Rev.  de  med., 
Paris,  1892. 

2  Ann.  de  Flnst.  Pasteur,  Paris,  1895,  t.  ix,  p.  593. 

3  La  Cellule,  Lierre  et  Louvaio,  1895,  t.  xi,  p.  175  ;  Bull  Acad.  roy.  de  med.  de 
Belg.,  Bruxelles,  1895,  No.  11. 

16—2 


244  Chapter  VIII 

of  fresh  un vaccinated  rabbits.  When,  however,  he  injected  cultures 
grown  in  the  serum  of  immunised  animals  into  rabbits,  these  rabbits 
did  not  die  and  presented  only  transient  and  insignificant  lesions. 
From  this  fact  Roger  concluded  that  there  must  be  an  attenuation 
of  the  streptococcus  by  the  immune  serum,  a  view  which  was  shared 
by  several  other  observers.  In  formulating  this  view,  however,  he 
had  not  taken  into  account  the  possibility  that  this  serum  acted  not 
upon  the  coccus  that  had  developed  in  it  but  upon  the  organism 
of  the  animal  into  which  it  was  injected.  Bordet1,  indeed,  was  able 
to  show  that  the  streptococcus  which  grows  in  the  serum  of  im- 
munised animals  is  in  no  way  weakened  in  virulence.  When  he 
took  a  race  very  virulent  for  the  rabbit  (Marmorek's  streptococcus) 
and  injected  a  minimal  dose  of  a  culture  grown  in  the  serum  of 
immunised  animals,  the  rabbits  died  just  as  did  the  control  animals, 
because  the  amount  of  serum  introduced  was  too  small  to  exert 
any  influence.  So  also,  when  he  filtered  this  culture  and  got  rid 
of  the  serum  bathing  the  streptococci,  it  was  found  to  be  just  as 
[257]  virulent  as  that  grown  in  the  serum  of  susceptible  unvaccinated 
animals. 

In  confirmation  of  the  discovery  made  by  Roger  with  the  serum 
of  vaccinated  rabbits,  Bordet  showed  that  the  blood  serum  of  horses 
highly  immunised  against  the  streptococcus  did  not  exhibit  any 
bactericidal  action.  Moreover,  he  found  that  this  serum  caused  the 
development  of  somewhat  agglutinated  streptococci  and  that  it  was 
capable  of  throwing  streptococci  grown  on  the  ordinary  media  into 
clumps.  Summing  up  his  researches  on  the  properties  of  this  serum 
Bordet  concludes  that  it  "  causes  no  profound  change  in  the  strepto- 
coccus. The  vegetative  character  of  the  coccus  is  not  appreciably 
diminished,  and  its  morphology  remains  the  same  save  for  certain 
variations  in  the  length  of  the  chains.  Even  the  agglutinative 
power,  recognised  in  numerous  serums  by  recent  researches,  is,  in 
the  antistreptococcic  serum,  developed  but  slightly "  (p.  196). 

More  recently  von  Lingelsheim2  has  studied  the  properties  of 
the  serum  of  animals  which  he  had  thoroughly  vaccinated  against 
the  streptococcus.  He  observed  a  certain  slowing  of  the  development 
of  the  coccus  in  this  serum  as  compared  with  the  growth  in  cultures 
made  in  the  serum  of  normal,  susceptible  animals.  But  this  re- 

1  Ann.  de  VInst.  Pasteur,  Paris,  1897,  t.  xi,  p.  194. 

2  Arch,  internal,  de  Pharmacodyn.,  Gand  et  Paris,  1899,  Vol.  vi,p.  73;  Behring's 
"  Beitr.  z.  experim.  Therapie,"  1899,  Bd.  I. 


Facts  bearing  on  acquired  immunity  245 

tardation  was  slight  and  transient,  and  exhibited  itself  especially 
in  serums  to  which  von  Lingelsheim,  following  Denys,  had  added 
leucocytes. 

Von  Lingelsheim  also  noted  a  certain  degree  of  agglutination 
of  the  streptococcus  by  the  serum  of  vaccinated  animals,  although 
this  was  much  more  feeble  than  in  the  case  of  the  cholera  vibrio 
or  the  typhoid  bacillus,  when  agglutinated  by  their  corresponding 
serums.  Speaking  generally,  he  regarded  the  direct  action  of  the 
body  fluids  as  insufficient  to  bring  about  the  rapid  destruction  of 
the  streptococci  in  the  vaccinated  organism.  "Since  the  action 
of  the  bactericidal  substances  is  limited  in  time,  the  streptococci 
are  able  to  adapt  themselves  to  these  substances  and  recover  their 
former  energy.  As  the  phenomena  of  extracellular  solution,  of  such 
a  form  as  those  observed  under  the  influence  of  the  cholera  anti- 
,  bodies,  are  absent  in  the  case  of  the  streptococcus  and  as,  on  the 
other  hand,  a  considerable  ingestion  of  these  organisms  by  the  leuco- 
cytes is  observed we  must  seek  in  the  activity  of  these  cells 

a  second  important  element  of  the  defence  of  the  animal  organism  "  ['258J 
(p.  78). 

To  Salimbeni1,  who  has  carried  out  in  my  laboratory  an  in- 
vestigation on  this  subject,  we  are  indebted  for  the  most  reliable 
information  on  the  phagocytic  reaction  in  acquired  immunity  against 
the  streptococcus.  He  studied  specially  the  phenomena  in  the  sub- 
cutaneous tissue  of  a  horse,  hypervaccinated  against  Marmorek's 
streptococcus  ;  this  animal  received  in  all,  at  several  injections,  about 
five  litres  of  living  culture.  In  spite  of  this  refractory  condition, 
an  oedema  at  the  point  of  inoculation  was  soon  produced ;  in  this 
the  micro-organisms  remained  free  and  the  leucocytes  were  sparse. 
But  the  cellular  reaction,  at  first  insignificant,  developed  with 
great  rapidity  and  many  leucocytes,  amongst  which  the  macro- 
phages  were  much  the  more  numerous,  were  attracted.  The  phago- 
cytosis was  still  delayed  for  some  time,  but  it  continued  to  increase 
and  20  to  24  hours  after  the  inoculation  it  was  complete.  As 
soon  as  the  phagocytosis  was  well  established  the  oedema  began 
to  disappear.  In  the  thick  exudation,  containing  a  mass  of  leuco- 
cytes, the  macrophages  are  filled  with  a  very  large  number  of 
streptococci  packed  together.  These  cocci  develop  inside  the  cells, 
cause  them  to  burst  and  again  become  free.  A  fresh  arrival  of 
leucocytes,  however,  takes  place,  this  time  mainly  microphages. 
1  Ann.  de  VInst.  Pasteur,  Paris,  1898,  t.  xn,  p.  192. 


246  Chapter  VIII 

These  microphages  seize  the  free  streptococci  that  have  struggled 
so  victoriously  against  the  macrophages ;  this  second  phagocytic 
phase  is  final.  The  streptococci  still  remain  alive  inside  the  micro- 
phages for  some  days,  but  ultimately  are  killed  and  digested  by 
the  phagocytes.  At  a  period  when,  5  or  6  days  after  injection, 
insignificant  or  isolated  traces  of  streptococci  are  to  be  found  in 
the  microphages,  the  exudation  when  sown  in  nutritive  media  still 
gives  abundant  cultures.  The  incidents  of  this  struggle  between  the 
streptococcus  and  the  animal  organism  demonstrate  the  important 
part  played  by  the  phagocytes.  The  fact  that  the  macrophages 
perish  and  allow  the  cocci  to  escape,  clearly  proves  that  these  cocci 
have  been  ingested  alive  and  virulent,  and  consequently  that  the 
fluid  of  the  exudation  was  incapable  of  destroying  or  even  of 
attenuating  them.  The  macrophages,  also,  were  powerless  to  bring 
[259]  about  this  result  and  the  intervention  of  the  microphages  was 
necessary  to  cause  the  disappearance  of  the  cocci.  It  is,  however, 
always  the  phagocytes  which  ensure  the  final  resistance  of  the 
animal. 

In  presence  of  these  very  precise  results  obtained  from  the  work 
of  Salimbeni,  a  work  which  I  followed  very  closely,  the  previous 
researches  by  Denys  and  Leclef  (l.c.)  made  under  less  favourable 
conditions  on  vaccinated  rabbits  are  deprived  of  their  importance. 
These  observers  wished  to  get  an  idea  of  the  difference  between  the 
reactions  of  the  animal  organism  (a)  after  the  injection  of  streptococci 
into  the  pleural  cavity  of  immunised  rabbits,  and  (b)  after  injection 
into  that  of  normal  susceptible  rabbits.  They  killed  the  inoculated 
animals  and  found  a  very  marked  diminution  of  micro-organisms  in 
the  pleuritic  exudation  of  the  former.  This  diminution  could  not  be 
attributed  to  a  lysis  of  the  streptococci  by  the  body  fluids,  because 
there  were  never  any  signs  of  such  destruction.  Nor  could  the 
phagocytosis,  very  feeble  at  first,  be  considered  as  the  cause  of  the 
disappearance  of  a  large  number  of  the  streptococci.  Denys  and 
Leclef  put  forward  a  third  hypothesis,  which  attributed  this  dis- 
appearance to  the  rapid  resorption  by  the  lymph  stream  of  the 
injected  fluid  containing  the  organisms.  Going  over  the  record  of 
their  experiments  it  will  be  seen  that  in  vaccinated  rabbits  the 
quantity  of  pleuritic  exudation  was  always  very  much  less  than  in 
normal  rabbits.  In  presence  of  this  feature  there  is  reason  to  ask 
whether,  in  the  case  of  the  streptococci,  a  large  number  of  these 
organisms  were  not  fixed,  along  with  the  leucocytes,  on  the  walls  of 


Facts  bearing  on  acquired  immunity  247 

the  pleura,  as  in  guinea-pigs  that  are  inoculated  intra-peritoneally  ? 
Instead  of  being  satisfied  with  merely  examining  the  fluid  exudation, 
the  surface  of  the  pleura  should  have  been  scraped  in  order  to  ascertain 
whether  the  phagocytic  reaction  was  localised  in  this  region. 

In  any  case  such  incomplete  results  on  the  active  immunity  of 
rabbits  in  no  way  weaken  the  positive  results  obtained  in  the  sub- 
cutaneous tissue  of  the  horse,  in  which  the  phagocytic  reaction  plays 
a  really  preponderant  part. 

This  example  of  the  streptococci  completes  our  series  of  bacteria 
in  which  we  have  studied  their  relations  with  the  properties  of  the 
animal  organism  that  has  acquired  immunity.  We  have  still  to  see 
whether  the  acquired  immunity  against  micro-organisms  of  animal 
origin  is  subject  to  the  same  law  as  that  against  bacteria. 

For  some  years  past  a  zealous  study  of  the  infectious  diseases  pro- 
duced by  animal  micro-organisms  has  been  carried  out.  Besides  [260] 
malaria,  which  occupies  a  most  important  position,  attention  has  been 
directed  to  certain  diseases  in  domestic  animals  that  are  set  up  by 
endoglobular  haematozoa  and  by  flagellata,  and  a  fairly  large  number 
of  accurate  data  have  been  collected  with  regard  to  Texas  fever  and 
its  parasite  the  Piroplasma  bigemhmm,  as  well  as  upon  the  epi- 
zootic diseases  due  to  Trypanosomata  (Tsetse  fly  disease  or  Nagana, 
"Dourine,"  etc.). 

We  are  indebted  to  Smith  and  Kilborne1  for  the  earliest  informa- 
tion concerning  the  acquired  immunity  of  Bovidae  against  Texas  fever. 
R  Koch2  then  added  some  very  precise  observations  on  the  immu- 
nity of  calves  which  had  been  inoculated  with  parasites  attenuated  in 
the  body  of  the  tick  (Boopliilus  bovis).  Lignieres3,  who  devoted 
much  attention  to  this  question  in  the  Argentine  Republic,  has  dis- 
covered a  sure  method  of  vaccinating  the  Bovidae  against  the 
"Tristeza,"  the  local  name  for  Texas  fever.  He  brought  to  Alfort 
specimens  of  attenuated  haematozoa,  and  in  Nocard's  presence 
performed  successful  vaccination  experiments.  Lignieres  is  now 
engaged  in  devising  a  practical  method  of  ensuring  immunity  under 
the  special  conditions  found  in  the  home  of  the  "Tristeza."  Up  to  the 
present,  however,  there  are  no  sufficient  data  as  to  the  mechanism  of 

1  Bulletin  No.  1,  Bureau  of  Animal  Industry,  U.S.  Dep.  of  Agric.,  Washington, 
1893. 

*  "  Reisebericht  iiber  Rinderpest  etc.,"  Berlin,  1898. 

3  Rec.  de  med.  vet.,  Paris,  juillet,  1900,  and  Ann.  de  Vlnst.  Pasteur,  Paris,  1901, 
t.  xv,  p.  121. 


248  Chapter  VIII 

the  acquired  immunity  in  this  case.  We  have  fuller  information  as  to 
the  essential  phenomena  observed  in  the  organism  of  the  rat  vacci- 
nated against  Trypanosoma  lewisi.  We  owe  to  Mine.  L.  Rabinowitsch 
and  Dr  Kempner1  the  first  important  data  as  to  the  possibility  of 
immunising  white  or  piebald  rats  against  the  disease  produced  by  the 
flagellated  infusorian.  They  noted  that  these  animals  when  inocu- 
lated with  the  blood  of  grey  rats  containing  Trypanosomata  acquire 
a  very  transitory  disease  which,  however,  confers  an  immunity  against 
any  subsequent  infection.  The  flagellated  organisms  disappear  from 
the  blood  within  a  few  weeks,  after  which  fresh  injections  of  these 
parasites  have  no  pathogenic  effect. 

[261]  Laveran  and  Mesnil2  confirmed  these  observations,  and  in  ad- 
dition made  careful  observations  on  the  mechanism  of  this  acquired 
immunity.  After  making  several  inoculations  with  blood  containing 
Trypanosomata  into  white  rats,  they  made  a  study  of  the  properties 
of  the  blood  serum  of  these  immunised  animals.  First  they  esta- 
blished the  fact  that  this  serum  exerts  no  microbicidal  action  on 
the  Trypanosomata,  but  it  agglutinates  them  without,  however, 
rendering  them  motionless: — "The  masses  may  be  resolved  into 
rosettes  in  which  the  Trypanosomata,  united  merely  by  their  poste- 
rior extremities,  have  their  flagella  free  and  motile  at  the  periphery." 
Laveran  and  Mesnil  then  studied  the  phenomena  evolved  in  the 
refractory  organism.  When  injected  into  the  peritoneal  cavity  of 
immunised  rats  the  Trypanosomata  are  not  acted  upon  injuriously  by 
the  body  fluids.  They  are,  however,  devoured  by  the  leucocytes. 
Laveran  and  Mesnil  thus  express  themselves  on  this  subject :  "...we 
have  demonstrated  clearly  and  repeatedly  that  the  Trypanosomata 
are  ingested  alive,  perfectly  isolated  and  very  motile,  by  phagocytes, 
and  we  have  followed  the  details  of  this  process  of  ingestion  which 
recalls  that  of  the  ingestion  of  spirilla  by  the  leucocytes  of  the 
guinea-pig.  We  consider,  therefore,  that  the  immunity  is  phagocytic 
in  character." 

The  main  facts  on  acquired  immunity  established  in  connection  with 
the  most  diverse  micro-organisms,  facts  just  described,  may  already  be 
said  to  lead  to  certain  general  conclusions.  They  indicate  in  the 
first  place  that  acquired  immunity  is  accompanied  by  phenomena 
more  complicated  than  those  observed  in  natural  immunity.  In  the 
two  categories  of  processes  observed  in  acquired  immunity  the  pha- 

1  Ztschr.f.  Hyg.,  Leipzig,  1899,  Bd.  xxx,  S.  251. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  673. 


Facts  bearing  on  acquired  immunity  249 

gocytic  reaction  is  the  only  one  that  can  be  said  to  be  constant.  We 
find  it  in  those  examples  in  which  the  influence  of  the  fluids  of  the 
body  is  most  manifest,  as  in  the  experimental  cholera  peritonitis  of 
the  guinea-pig,  as  well  as  in  those  cases  where  the  humoral  action  is 
most  feeble,  as  in  anthrax  or  in  the  Trypanosoma  disease  of  rats. 
\Ve  have,  however,  still  to  establish  the  relations  that  exist  between 
phagocytosis  and  the  part  played  by  the  fluids  of  the  immunised 
animal,  in  order  that  we  may,  as  far  as  possible,  present  a  general 
picture  of  the  inner  mechanism  of  acquired  immunity  against  micro- 
organisms. To  attain  this  result  we  must  place  the  reader  in  posses-  [262] 
sion  of  further  well-established  facts,  and  we  must  postpone  its 
discussion  to  the  following  chapter,  which  will  be  entirely  devoted 
to  the  above-mentioned  problem. 


[263]  CHAPTER  IX 

THE  MECHANISM  OF  ACQUIRED  IMMUNITY  AGAINST 
MICRO-ORGANISMS 

Cytases  and  fixatives. — Only  the  latter  are  augmented  in  the  immunised  organism. — 
Properties  of  the  fixatives. — Difference  between  them  and  the  agglutinative 
substances. — The  part  played  by  the  latter  in  acquired  immunity. — Protective 
property  of  the  fluids  of  the  immunised  organism. — Stimulant  action  of  the  body 
fluids. — The  protective  power  of  serum  cannot  serve  as  a  measure  of  acquired 
immunity. — Examples  of  acquired  immunity  in  which  the  serums  exhibit  no 
protective  power. — Phagocytosis  in  acquired  immunity. — Negative  chemiotaxis 
of  leucocytes. — Theory  of  attenuation  of  micro-organisms  by  the  fluids  of  im- 
munised animals. — Refutation  of  this  theory. — Phagocytosis  acts  without  requiring 
any  previous  neutralisation  of  the  toxins. — The  origin  of  the  fixative  and  pro- 
tective properties  of  the  body  fluids. — The  relation  between  these  properties  and 
phagocytosis. — The  side-chain  theory  of  Ehrlich  and  the  theory  of  phagocytes. 

WHILST,  in  natural  immunity  against  micro-organisms,  humoral 
phenomena  play  no  prominent  part,  in  acquired  immunity  these 
phenomena  assume  a  much  greater  importance.  The  bactericidal 
power  of  the  fluids  of  the  body  is,  in  natural  immunity,  reduced  to  a 
mere  trace,  for  it  has  been  demonstrated  that  the  power  of  normal 
serums  to  destroy  bacteria  corresponds  to  no  natural  phenomenon  of 
the  living  organism,  but  is  dependent  upon  the  presence  of  cytases 
which  have  escaped  from  the  phagocytes  at  the  time  of  the  formation 
of  the  clot  in  vitro  and  separation  of  the  serum.  The  presence  of  the 
fixative,  that  other  important  element  in  immunity,  has  been  demon- 
strated in  the  normal  fluids  only  in  rare  cases  and  in  small  quantity. 
The  agglutinative  property  of  these  fluids  has  likewise  shown  itself 
to  be  little  developed  and  without  any  importance  in  natural 
immunity. 

In  acquired   immunity  against    micro-organisms,    on  the   other 
hand,  we  find  that  the  bactericidal  and  agglutinative  powers  of  the 


Acquired  immunity  against  micro-organisms      251 

fluids  of  the  body  are  very  greatly  increased.  With  the  discovery  that 
the  bactericidal  property  was  so  highly  developed  in  the  serums  of 
animals  that  had  been  vaccinated  against  vibrios  arose  the  belief  in  [264] 
the  acquisition  of  a  new  and  purely  humoral  property.  R.  Pfeiffer, 
especially,  insisted  on  the  fundamental  difference  between  the  power 
of  the  serum  of  immunised  animals  to  transform  the  cholera  vibrios 
into  granules  and  the  corresponding  property  of  normal  serums.  In 
the  first  case  Pfeiffer's  phenomenon  exhibited  marked  specificity  ; 
in  the  second,  it  was  much  more  general.  A  normal  serum  trans- 
forms into  granules,  indifferently,  vibrios  that  are  very  distinct  from 
one  another ;  whilst  the  serum  of  an  animal  vaccinated  against 
a  particular  species  or  race  of  vibrios  gives  Pfeiffer's  phenomenon 
with  this  species  or  race  only.  Bordet's1  researches  have  defi- 
nitely settled  this  question.  This  investigator  has  shown  that 
Pfeiffer's  phenomenon  is  produced,  with  all  the  usual  serums,  by 
means  of  the  same  substances,  the  cytases  (alexine,  or  complement  of 
Ehrlich).  But  in  the  serum  of  vaccinated  animals  there  is  added 
to  these  cytases  the  fixative  (sensibilising  substance  of  Bordet, 
immunising  body  or  amboceptor  of  Ehrlich)  which  exhibits  specific 
properties.  Having  thus  carefully  distinguished  the  two  substances 
that  set  up  the  granular  change  in  vibrios,  Bordet  shows  that  in 
vaccinated  animals  it  is  the  fixative  which  increases  in  quantity, 
whilst  the  cytase  remains  pretty  much  in  the  same  proportions  as  in 
the  normal  animal.  He  demonstrated,  in  fact,  that  when  we  take 
a  very  small  dose  of  the  serum  of  a  vaccinated  animal  which  by  itself 
is  incapable  of  transforming  the  vibrios  into  granules,  about  the  same 
quantity  of  immunised  serum  or  of  normal  serum  must  be  added 
to  it  in  order  that  Pfeiffer's  phenomenon  may  appear.  The  quantity 
of  cytase,  that  soluble  ferment  which  is  necessary  for  the  production 
of  the  phenomenon,  is,  therefore,  about  the  same  in  the  serum  of 
a  normal  animal  as  in  that  of  a  well-vaccinated  animal.  Whilst  the 
cytase  does  not  increase  as  a  result  of  vaccinal  injections,  the  fixative, 
•  on  the  other  hand,  becomes  more  and  more  abundant.  Consequently 
it  is  this  second  soluble  ferment  that  impresses  its  characters  on  the 
blood  serum  and  on  some  of  the  other  fluids  of  the  vaccinated  animal. 
It  has  been  pointed  out  in  the  preceding  chapter  that  the  fixative  is 
found  in  the  fluid  of  the  oedema  of  vaccinated  animals,  although  in 
less  quantity  than  in  their  blood  serum.  It  has  also  been  mentioned 
that  no  fixative  is  found  in  the  aqueous  humour  of  well-vaccinated  [265] 
1  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  462. 


252  Chapter  IX 

animals.  It  must  be  admitted  that  this  ferment  is  not  inseparably 
bound  to  the  cells  which  produce  it,  as  is  the  case  with  the  cytases. 
I  have  already  developed,  at  some  length,  the  thesis  that  the  cytases 
remain,  in  the  normal  animal,  within  the  phagocytes,  and  only  escape 
from  them  when  these  cells  are  destroyed,  whether  in  the  living 
animal  (during  phagolysis)  or  outside  the  animal  (during  the  prepa- 
ration of  the  serum).  Gengou's  experiments  with  the  plasma  and 
the  blood  serum  of  normal  animals  have  completely  confirmed  the 
fundamental  observations  that  the  cytases  are  not  found  free  in  the 
circulating  blood.  It  is  evident  that  the  same  law  applies  also  to 
an  animal  that  has  acquired  immunity.  For  this  reason  neither 
Pfeiffer's  phenomenon  nor  any  analogous  process  that  demands  the 
action  of  cytases  is  ever  produced  in  the  anterior  chamber  of  the 
eye,  or  in  the  subcutaneous  tissue,  or  in  oedema  either  active  or 
passive.  Further,  it  is  in  virtue  of  this  same  law  that  Pfeiffer's 
phenomenon  does  not  manifest  itself  even  in  the  peritoneal  cavity  or 
in  the  blood  vessels  of  vaccinated  animals  in  which  the  phagocytes 
have  been  protected  from  phagolysis  by  previous  injections  of 
various  fluids  (physiological  saline  solution,  broth,  etc.).  It  would 
be  very  interesting  to  be  able  to  demonstrate  the  absence  of  cytases 
in  the  fluids  of  immunised  animals  by  experiments  of  the  same  order 
as  those  carried  out  by  Gengou  with  the  fluids  of  normal  animals, 
but  the  obstacles  to  the  realisation  of  this  postulate  are  too  great. 
We  saw  when  discussing  Gengou's  experiments  that  it  is  impossible 
to  obtain  in  vitro  a  fluid  identical  with  the  plasma  of  living  blood. 
The  greatest  precautions  in  collecting  the  blood  and  in  its  after 
treatment  are  insufficient  to  prevent  coagulation  taking  place  sooner 
or  later.  It  follows  that,  as  there  is  always  a  considerable  quantity 
of  free  fixative  in  the  plasma  of  immunised  animals,  an  infinitesimal 
quantity  of  microcytase,  set  free  from  the  leucocytes,  is  sufficient 
for  the  production  of  Pfeiifer's  or  any  other  analogous  phenomenon. 
There  must  be  a  great  improvement  in  the  methods  of  preparation 
of  plasmas  outside  the  body  before  it  will  be  possible  to  undertake 
successful  researches  on  the  above  problem.  For  the  present  we 
must  rest  satisfied  with  other  proofs,  already  numerous  and  very 
demonstrative,  of  the  absence  of  free  cytases  in  the  normal  plasmas  of 
vaccinated  animals. 

[266]  The  cytases  being  found  in  about  the  same  quantity  and  pre- 
senting the  same  properties  in  all  animals  that  enjoy  immunity 
whether  natural  or  acquired,  it  must  be  the  fixative  which  specially 


Acquired  immunity  against  micro-organisms      253 

distinguishes  these  two  categories  of  immunity.  Xow,  the  fixative 
is  found  in  the  serum  of  perhaps  all  cases  of  acquired  immunity. 
Bordet  and  Gengou  have  studied  it  by  the  method  already  mentioned 
(Chap.  VII.) .  A  certain  quantity  of  micro-organisms  of  various  species 
is  introduced  into  the  serum.  If  the  cytases,  present  in  the  serum 
when  the  experiment  was  commenced,  ultimately  disappear  from  it, 
it  indicates  that  this  ferment  has  been  absorbed  by  the  bacteria, 
thanks  to  the  fixative,  which  consequently  should  be  found  in  the 
serum  under  observation.  The  presence  or  absence  of  the  cytases 
can  be  demonstrated  by  the  production  or  absence  of  Pfeiffer's  phe- 
nomenon with  vibrios. 

The  application  of  this  method  enabled  Bordet  and  Gengou1 
to  satisfy  themselves  that  the  serum  of  animals  immunised  against 
several  species  of  bacteria  (plague  bacillus,  typhoid  bacillus,  bacillus 
of  swine  erysipelas,  first  anthrax  vaccine,  and  Proteus  vulgdris),  really 
contains  an  appreciable  quantity  of  fixative.  It  may,  then,  be  ac- 
cepted that  the  production  of  this  substance  is  fairly  constant  in 
acquired  immunity  against  bacteria,  and  that  it  constitutes  one  of  the 
most  important  factors  in  such  immunity. 

The  question  has  been  raised  :  What  is  the  nature  of  the  substance 
to  which  the  name  of  fixative  is  given?  Pfeifier  and  Proskauer2 
have  attempted  to  solve  this  question  by  making  use  of  a  serum  which 
acts  against  the  cholera  vibrio  and  which  they  obtained  by  vaccinating 
animals  with  this  vibrio.  They  carried  out  a  long  series  of  experi- 
ments which  led  them  to  the  conclusion  that  this  substance,  which 
they  term  "  cholera  antibody,"  cannot  be  identified  with  any  of  the 
albuminoid  substances  of  the  serum.  Further,  the  fixative  is  not 
represented  by  any  of  the  salts  or  extractive  substances  of  the  serum, 
because  these  substances  dialyse  easily,  whereas  the  cholera  antibody 
does  not  pass  through  the  dialysing  membrane.  The  fixative  is  wholly 
precipitated  by  alcohol,  and  is  regarded  by  Pfeiffer  and  Proskauer  as 
belonging  to  the  category  of  soluble  ferments,  an  opinion  which  is 
certainly  shared  by  many  other  investigators. 

What  is  it  that  communicates  to  this   ferment  its   remarkably  [267] 
specific  character?     Without  being  able  to  give  a  precise  answer 
to  this  question,  the  authors  just  cited  point  out  the  analogy  that 
exists  between  the  cholera  antibody  and  the  soluble  ferments  of 
yeasts  which  have  been  studied  by  Emil  Fischer.    Some  of  these 

1  Ann.  de  Vlnst.  Pasteur,  Paris,  1901,  t  xv,  p.  289. 

2  CentralU.f.  Bacteriol.  u.  Parasitenk.,  Jena,  1896,  lte  Abt,  Bd.  xix,  S.  191. 


254  Chapter  IX 

act  only  upon  certain  special  sugars  in  a  manner  equally  specific. 
From  a  logical  point  of  view  it  might  be  permissible  to  attribute 
the  specificity  of  fixatives  to  something  borrowed  from  the  species 
of  micro-organism  that  has  played  a  part  in  their  production.  It 
has  long  been  recognised  that  in  old  cultures  of  the  cholera  vibrio 
these  micro-organisms  are  transformed  into  spherical  granules,  the 
arthrospores  of  Hueppe,  which  closely  resemble  the  granules  produced 
in  PfeifFer's  phenomenon.  There  are,  then,  undoubtedly,  vibrionic 
products  which  act  much  as  do  the  microcytases,  and  it  would  be  very 
interesting  if  we  could  find  them  in  the  bactericidal  ferments  of  the 
animal  body.  An  attempt  of  this  kind  was  undertaken  by  Emmerich 
and  Low1,  who  attribute  the  acquired  immunity  to  a  particular  sub- 
stance which  they  term  "  Nuclease-Immunprotei'din."  According  to 
their  hypothesis  the  microbial  products  which  are  produced  in  the 
animal  during  the  period  of  vaccination — the  nucleases — combine  with 
proteid  substances  of  the  blood  and  organs  to  furnish  the  substance 
to  which  these  authors  have  given  such  an  elaborate  name.  In  their 
most  recent  publication  Emmerich  and  Low  even  describe  a  method 
of  producing  this  substance  outside  the  animal  body,  by  the  action 
of  ox  blood,  or  better  still  pounded  spleen,  on  the  nuclease  produced 
by  the  bacteria  found  in  old  cultures.  To  it  they  attribute  the  pro- 
perty of  dissolving  the  various  bacteria,  of  conferring  immunity 
against  and  even  of  curing  several  infective  diseases.  But  these 
authors  do  not  say  whether  this  remarkable  substance  is  identical 
with,  or  analogous  to,  the  antimicrobial  ferments  composed,  as  we 
have  seen,  of  microcytase  and  fixative.  It  must  be  concluded  that 
they  look  upon  it  as  being  similar  to  the  alexine  of  Buchner,  which 
is  nothing  more  than  a  mixture  of  the  two  substances  just  named. 
Unfortunately  the  whole  account  given  by  Emmerich  and  Low  will 
do  anything  but  gain  over  the  reader,  and  in  their  publications  no 
proof  of  their  assertions  can  be  found.  Several  of  the  facts  advanced 
by  them  do  not  fall  in  with  well-established  data.  Thus  they  speak 
[268]  of  the  complete  lysis  of  the  bacilli  of  swine  erysipelas  by  their 
soluble  "  Erysipelase-Immunprotei'din  "  in  vaccinated  animals,  a  pro- 
cess that  has  never  been  demonstrated  by  them  and  which  in  no 
way  accords  with  conscientious  and  carefully  carried  out  observations. 
On  the  other  hand,  they  cite  facts  which  contradict  one  another.  The 
"  Pyocyanase-Immunproteidin "  is  a  substance  which  possesses  an 
extraordinary  bactericidal  power,  not  only  against  the  Bacillus  pyo- 
1  Ztschr.f.  Hyg.,  Leipzig,  1901,  Bd.  xxxvr,  S.  9. 


Acquired  immunity  against  micro-organisms      255 

cyaneus  but  also  against  several  other  bacteria,  e.g.  the  bacilli  of 
anthrax,  diphtheria,  typhoid,  and  plague.  This  substance  rapidly 
breaks  up  these  bacteria,  and  cures  diphtheria  and  experimental 
anthrax.  But  it  is,  at  the  same  time,  so  affected  by  the  invasion 
of  the  most  common  bacteria,  such  as  Bacillus  subtilis,  that  it  is 
necessary  to  add  antiseptics  in  order  to  preserve  it  To  these  con- 
tradictions, inaccuracies,  and  uncertainties  must  be  added  further 
the  advice,  given  by  Emmerich  and  Low  to  bacteriologists,  not  to 
attempt  to  reproduce  their  experiments,  because  they  may  easily 
fail,  and  I  think  that,  in  spite  of  the  seductiveness  of  the  attempt 
to  attribute  to  bacterial  products  a  share  in  the  elaboration  of  anti- 
microbial substances,  we  must  conclude  not  to  follow  these  authors 
further.  It  is  better  to  confess  our  ignorance  of  the  chemical 
composition  of  these  substances  in  general  and  of  the  fixatives  in 
particular. 

As  the  fixatives  resist  temperatures  much  higher  than  those  which 
destroy  the  cytases,  in  this  respect  resembling  the  agglutinative  sub- 
stances so  frequently  found  in  the  fluids  of  vaccinated  animals,  there 
has  long  been  a  tendency  to  identify  them  with  these  latter.  It  is  in- 
disputable that  between  the  fixatives  and  the  agglutinative  substances 
the  analogies  are  fairly  numerous.  Both  are  produced  in  quantity 
during  the  process  of  immunisation,  and  are  found  not  only  in  the 
blood  serum  but  also  in  the  fluids  of  the  living  animal,  especially 
in  the  fluids  of  exudations  and  transudations.  Both  dialyse  through 
parchment  more  readily  than  do  the  cytases.  Buchner1  has  demon- 
strated that  his  alexiues  (bactericidal  substances  of  normal  serum) 
will  dialyse  only  where  the  lower  fluid  is  pure  water ;  dialysis  is  nil 
when  the  distilled  water  is  replaced  by  physiological  saline  solution. 
The  fixatives  and  agglutinins,  as  demonstrated  by  Gengou2  for  the  [269] 
latter,  pass  almost  completely  through  the  dialyser  in  the  case  of  pure 
water,  and  one-half  still  passes  when  the  lower  fluid  approaches  as 
nearly  as  possible  to  normal  serum. 

In  spite  of  these  analogies,  however,  the  agglutinative  property 
must  be  sharply  distinguished  from  the  fixative  power  of  serums.  In 
this  fluid,  derived  from  normal  animals,  the  agglutinative  property  is 
often  very  marked  when  the  power  of  fixing  the  cytases  is  totally,  or 
in  great  part  absent.  Bordet  and  Geugou3  have  demonstrated  also 

1  Munchen.  med.  Wchnschr.,  1892,  SS.  119,  982. 

2  Ann,  de  Flnst.  Pasteur,  Paris,  1899,  t  xin,  p.  647. 

3  Ann.  de  Flnst.  Pasteur,  Paris,  1901,  t.  xv,  p.  289. 


256  Chapter  IX 

that  feebly  agglutinative  serums  of  persons  convalescent  from  typhoid 
fever  may  exhibit  a  great  capacity  for  fixing  the  cytases.  Other 
facts,  to  be  mentioned  later,  confirm  the  real  difference  between  the 
fixative  and  the  agglutinative  properties. 

The  agglutination  of  bacteria  was  noted  during  the  course  of  a 
series  of  researches  on  the  acquired  properties  of  the  blood  serum  of 
vaccinated  animals.  Charrin  and  Roger1,  seeking  to  obtain  a  clear 
idea  of  the  difference  between  the  serum  of  normal  animals  and  that 
of  animals  vaccinated  against  the  Bacillus  pyocyaneus,  observed  that 
this  bacillus  developed  in  the  normal  fashion  in  the  former,  but  in 
the  latter  gave  rise  to  special  forms  of  growth.  Instead  of  growing 
in  the  form  of  rods,  it  elongates  into  segmented  filaments  which 
become  entangled  and  fall  to  the  bottom  of  the  tubes,  leaving  a 
supernatant  limpid  serum.  I  was  able  not  only  to  confirm  the 
accuracy  of  this  observation  for  the  Bacillus  pyocyaneus,  but  to 
extend  it  to  Gamaleia's  vibrio  and  to  the  pneumococcus2.  In  all 
these  instances  we  have  a  modification  of  the  bacteria  developed 
in  specific  serums  coming  from  vaccinated  animals.  Later,  Bordet3, 
during  his  researches  on  the  bacteriolysis  of  vibrios  in  vitro,  observed 
that  these  vibrios,  when  introduced  into  the  blood  serum  of  vacci- 
nated animals,  lose  their  movements  and  soon  unite  into  more  or 
less  voluminous  masses.  This  observation  was  confirmed  by  Gruber 
[270]  and  Durham4,  who  were  the  first  to  apply  it  in  the  specific  diagnosis 
of  bacteria.  They  showed  that  the  agglutinating  power  of  vacci- 
nated animals,  although  not  rigorously  specific,  might,  nevertheless, 
be  utilised  for  the  differentiation  of  certain  bacteria,  especially  the 
cholera  vibrio  and  the  typhoid  bacillus.  But,  independently  of 
this  result,  Gruber5  essayed  to  formulate  a  theory  of  acquired  im- 
munity based  on  the  agglutinative  property  of  the  serum.  He 
accepted,  in  connection  with  the  phenomenon  of  the  destruction  of 
the  bacteria,  Bordet's  hypothesis  of  the  concurrent  action  of  two 
substances,  of  which  one,  the  bactericidal  substance  proper,  is  nothing 
but  the  alexine  of  Buchner,  the  second  being  that  which  agglutinates 
the  bacteria.  This  agglutination,  according  to  Gruber,  results  from 

1  Compt.  Rend.  Soc.  de  Biol.,  Paris,  1889,  p.  667. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1891,  t.  v,  p.  473. 

3  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  462. 

4  Munchen.  med.  Wchnschr.,  1896,  S.  285  [cf.  also  Durham,  Journ.  Path,  and 
Bacterial.,  Edin.  and  London,  1897,  Vol.  rv,  p.  13,  and  1901,  Vol.  vn,  p.  240;  Brit. 
Med.  Journ.,  London,  1898,  Vol.  n,  p.  588]. 

6   Wien.  klin.  Wchnschr.,  1896,  SS.  183,  204. 


Acquired  immunity  against  micro-organisms      257 

the  swelling  of  the  bacterial  membrane  which  becomes  viscous  and  so 
leads  to  the  cohesion  of  the  bacteria  and  the  formation  of  clumps. 
Thus  transformed  and  rendered  motionless,  the  bacteria  succumb  more 
readily  to  the  destructive  action  of  the  alexine.  It  is  supposed  that 
the  phagocytes  do  not  intervene  at  all  in  these  cases  of  acquired 
immunity,  except  in  a  purely  secondary  fashion  when  they  ingest 
the  bacteria  already  greatly  weakened  by  the  united  action  of  the 
agglutinm  and  the  alexine.  The  principal  rdle  in  this  theory  of 
immunity  is  thus  given  to  the  agglutinative  substance,  which  is  re- 
garded as  being  a  microbial  product,  modified  by  the  macrophages 
and  thrown  into  the  blood. 

The  discovery  of  this  agglutination  of  bacteria  has  acquired  great 
importance,  especially  in  connection  with  its  application  to  the  dia- 
gnosis of  typhoid  fever.  Widal1  succeeded  in  showing  that  typhoid 
bacilli  agglutinate  readily  under  the  influence  of  blood  serum  and 
other  fluids  (milk,  transudations,  tears,  etc.)  derived  from  patients 
suffering  from  typhoid  fever.  As  this  phenomenon  could  be  utilised 
for  the  early  recognition  of  the  disease,  it  began  to  be  studied  with 
great  care  and  many  interesting  data  concerning  it  have  been  col- 
lected. The  general  outcome  of  these  researches  accords  with  the 
conclusions  drawn  by  Widal,  and  the  serum-diagnosis  of  typhoid 
fever  has  taken  an  important  place  among  the  methods  used  for 
the  recognition  of  this  disease.  This  aspect  of  the  question,  however, 
does  not  interest  us  from  the  point  of  view  of  the  problem  of  im- 
munity which  we  now  have  under  consideration,  and  we  cannot  here 
enter  upon  the  study  of  the  serum-diagnosis  of  typhoid  fever  and 
certain  other  diseases  (cholera,  tuberculosis,  pneumonia).  Moreover,  [271] 
we  must  refrain  from  any  analysis  of  the  hypotheses  advanced  to 
explain  the  mechanism  of  agglutination.  A  lively  discussion  has  been 
carried  on  between  the  partisans  of  the  chemical  theory — according 
to  whom  the  agglutinin  acts  directly  on  the  agglutinable  substance 
of  the  bacteria — and  the  advocates  of  the  physical  theory,  led  by 
Bordet2,  who  attribute  the  agglutination  to  modifications  in  the 
molecular  attractions  which  unite  the  agglutinable  elements,  be  it 
between  each  other  or  with  the  surrounding  fluid.  At  one  time 
it  was  thought  that  Roger's3  observation  that  the  cell  membranes 
of  Outturn  albicans,  when  cultivated  in  the  specific  serum  of 

1  Bull.  Sac.  med.  d.  hop.,  Paris,  1896,  26  juin  [Semaine  med.t  Paris,  1896,  p.  259]. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1899,  t.  xm,  p.  225. 

3  Rev.. gen.  d.  sc.  pures  et  appliq.,  Paris,  1896,  t.  vii,  p.  770. 

B.  17 


258  Chapter  IX 

immunised  animals,  increased  in  volume  and  became  greatly  swollen, 
settled  the  question  in  favour  of  Gruber's  theory.  But  the  objection 
formulated  by  Kraus  and  Seng1,  on  the  one  hand,  and  by  Bordet, 
on  the  other,  dealt  a  severe  blow  to  this  view.  As  the  serum  em- 
ployed by  Roger  was  not  deprived  of  its  cytases  (alexine),  the 
viscosity  of  the  membrane  of  the  fungus  could  not  be  attributed 
to  the  agglutinin.  When  Bordet2  demonstrated  that  the  red  blood 
corpuscles,  under  the  influence  of  the  serums,  undergo  an  agglutina- 
tion as  marked  as  that  seen  in  bacteria,  it  enabled  us  to  study  this 
phenomenon  in  the  large  red  corpuscles  of  birds,  in  which  no  one 
has  ever  been  able  to  demonstrate  any  viscosity  of  the  corpuscular 
stroma.  In  a  mixture  of  red  corpuscles  of  bird  and  mammal,  sub- 
mitted to  the  action  of  a  serum  which  agglutinates  the  former  only, 
the  red  corpuscles  of  the  mammal  never  unite  with  those  of  the  bird, 
although  this  should  undoubtedly  take  place  if  the  membrane  of  the 
agglutinated  corpuscles  had  really  become  viscous.  All  the  facts 
collected  up  to  the  present  are,  therefore,  in  favour  of  Bordet's 
physical  theory  in  which  an  analogy  between  the  phenomena  of 
agglutination  and  of  coagulation  is  traced. 

The  point  that  interests  us  more  particularly  in  regard  to  aggluti- 
nation is  the  relation  of  this  phenomenon  to  immunity.  We  have 
already  given  (Chapter  VII)  the  arguments  which  render  it  impossible 
for  us  to  attribute  to  the  agglutinative  property  of  the  fluids  of  the 
body  any  rdle,  however  unimportant,  in  natural  immunity  against 
[272]  micro-organisms.  We  must  now  study  the  importance  of  this 
property  in  the  condition  of  acquired  immunity,  in  which  the  agglu- 
tination of  micro-organisms  by  the  fluids  of  the  body  is  much  more 
frequent  and  active  than  in  natural  immunity. 

The  first  question  to  be  settled  is  the  following:  Is  the  aggluti- 
native property  really  constantly  present  in  the  fluids  of  vaccinated 
animals  ?  The  blood  serum  of  animals  that  have  acquired  immunity 
is  unquestionably  usually  agglutinative  as  regards  the  corresponding 
micro-organism.  This  agglutination  may  be  more  or  less  pronounced, 
but  it  certainly  exists  in  the  great  majority  of  cases.  Nevertheless, 
examples  can  be  cited  in  which,  in  spite  of  the  refractory  condition 
acquired  as  the  result  of  immunisation,  the  serum  exhibits  not 
a  trace  of  agglutinative  power.  Having  demonstrated  that  several 
bacteria  (Bacillus  pyocyanms,  Diplococcus  pneumoniae,  Vibrio 

1  Wien.  klin.  Wchnschr.,  1899,  S.  1. 

2  Ann.  de  FInst.  Pasteur,  Paris,  1898,  t.  xn,  p.  688. 


Acquired  immunity  against  micro-organisms      259 

metchnttovi)  develop  in  the  serum  of  vaccinated  animals  in  the 
form  of  elongated  filaments  more  or  less  interlaced,  I  was  quite 
prepared  to  allow  that  this  fact  might  be  of  general  import.  But 
the  study  of  a  cocco-bacillus  which  produces  the  pueumo-enteritis  of 
swine  and  which  was  isolated  by  Chantemesse  during  an  epizootic 
at  Gentilly,  led  me  to  believe  that  this  was  not  the  case.  As  this 
bacillus  is  characterised  by  great  motility,  I  concluded1  that  it 
was  identical  with  that  of  the  hog  cholera  of  American  writers. 
Theobald  Smith2,  to  whom  I  sent  a  specimen  and  who  is  a  competent 
authority  on  this  question,  refers  it,  however,  to  the  species  which 
produces  swine  plague.  Knowing  that  the  question  of  these  two 
bacteria  is  not  finally  settled,  it  is  impossible  to  come  to  an  absolute 
decision  in  the  matter.  Fortunately,  from  the  point  of  view  of 
immunity,  this  is  of  no  great  importance.  The  point  upon  which 
I  must  lay  stress  is  that  the  serum  of  rabbits  vaccinated  against 
the  Gentilly  bacillus,  when  sown  with  this  cocco-bacillus,  gave  very 
abundant  and  uniformly  turbid  growths.  In  my  researches,  under- 
taken at  a  period  when  the  rapid  agglutination  of  micro-organisms 
added  directly  to  the  specific  serum  had  not  yet  been  recognised, 
I  noted  merely  that  the  cocco-bacilli  which  grew  in  the  blood  serum 
of  vaccinated  rabbits  presented  their  normal  form  and  gave  rise  to 
a  general  turbidity  of  the  fluid.  Since  then,  however,  it  has  often 
been  observed  that  the  mode  of  development  of  a  micro-organism 
in  a  serum  gives  an  even  more  delicate  indication  than  does  the 
agglutination  properly  so  called,  produced  by  the  serum  to  which  [273] 
has  been  added  an  organism  cultivated  on  its  usual  medium.  Thus 
Pfaundler3  saw  that  Bacillus  coli  and  Proteus  vulgaris,  which  were 
not  agglutinated  by  certain  serums,  developed  in  them  in  an  unusual 
fashion  and  produced  very  long  and  interlacing  filaments.  When 
a  serum  is  incapable  of  revealing  its  properties  by  agglutinative 
reaction  properly  so  called,  it  is  sown  with  the  corresponding  micro- 
organism and  the  development  is  then  compared  with  that  observed 
'  in  a  normal  serum.  Frequently  a  very  marked  difference  is  noted, 
the  same  organism  growing  into  filaments  in  the  specific  serum  and 
forming  rods  only  in  the  normal  serum.  The  first  mode  of  develop- 
ment is  sometimes  designated  "  Pfaundler's  reaction." 

1  Ann.  de  Hnst.  Pasteur,  Paris,  1892,  t.  vi,  p.  289. 

2  Centralbl.f.  Bakteriol.  u.  Parasitenk.,  Jena,  1894,  Bd.  xvi,  S.  235. 

8  Centralbl.f.  Bakteriol  u.  Parasitenk.,  Jena.  Ito  Abt.,  1898,  Bd.  xxm,  SS.  9,  71, 
131. 

17—2 


260  Chapter  IX 

In  the  serum  of  rabbits  vaccinated  against  the  Gentilly  cocco- 
bacillus,  no  filaments  corresponding  to  those  met  with  in  the  agglu- 
tinative reaction  are  formed,  but  bacilli  are  produced.  In  spite  of 
this  the  animals  that  furnish  the  serum  show  a  distinct  resistance 
to  infection.  More  recently,  Karlinski1  has  studied  the  properties  of 
the  serums  of  animals  treated  with  the  cocco-bacilli  of  hog  cholera 
and  swine  plague.  He  was  able  to  demonstrate  that  blood  serum 
from  oxen  that  had  received  repeated  injections  of  cultures  or  toxin 
of  hog  cholera,  was  not  only  incapable  of  killing  the  cocco-bacilli  of 
the  two  swine  diseases  but  it  even  "  gave  rise  to  no  agglutination  "  of 
the  two  bacilli  and  did  not  arrest  the  motions  of  those  of  hog  cholera. 
On  the  other  hand,  serums  have  been  obtained  from  other  species 
of  animals  (dog,  pig)  which  brought  about  the  typical  agglutination 
of  the  cocco-bacillus  of  hog  cholera2. 

In  the  preceding  chapter,  Gengou's  experiment  on  the  serum  of 
a  dog  that  had  been  treated  with  a  virulent  culture  of  anthrax  has 
already  been  cited.  This  serum  did  not  agglutinate  the  bacillus, 
even  of  the  first  vaccine  of  Pasteur.  Nevertheless,  a  second  dog 
treated  with  an  attenuated  culture  of  this  bacillus  furnished  an 
agglutinative  serum.  The  immunisation  of  the  first  dog  was  carried 
very  much  further  than  that  of  the  second,  but  the  agglutinative 
properties  were  in  inverse  order.  Sawtchenko,  in  his  study  of  im- 
munity against  anthrax,  demonstrated  that  the  subcutaneous  exu- 
[274]  dation  from  vaccinated  rats  does  not  agglutinate  the  bacillus  which 
usually  exhibits  such  a  great  tendency  to  collect  into  clumps. 

Agglutination  has  been  studied  particularly  carefully  in  typhoid 
fever.  We  know  that  after  an  attack  of  this  disease,  an  acquired 
refractory  condition  is  produced  which  lasts  for  a  considerable  period. 
In  most  cases  the  agglutinative  power  of  the  blood  diminishes  very 
rapidly,  and  disappears  a  few  weeks  after  the  commencement  of 
convalescence.  It  is  only  in  rare  cases  that  it  persists  for  years3. 
On  the  other  hand,  during  the  period  of  apyrexia  which  precedes 
the  relapse  in  typhoid  fever  and  during  the  period  of  relapse, 
the  agglutinative  power  may  manifest  itself  in  a  very  marked 
degree.  In  an  observation  made  on  a  case  reported  by  Widal  and 

1  Ztschr.f.  Hyg.,  Leipzig,  1898,  Bd.  xxvm,  S.  406. 

2  Fifteenth  Ann.  Rep.  of  the  Bureau  of  Animal  Industry  for  1898,  Washington, 
1899,  p.  348,  PI.  XI. 

3  Widal  et  Sicard,  Bull,  et  Mem.  Soc.  meet.  d.  hop.,  Paris,  1896,  p.  684  [Semaine 
med.,  Paris,  1896,  p.  514], 


Acquired  immunity  against  micro-organisms      261 

Sicard1,  the  agglutinative  power  was  raised,  two  days  before  the 
relapse,  to  a  ratio  (1  :  150)  it  had  never  attained  during  the  first 
attack.  "The  appearance  of  the  relapse,  two  days  after  this  ob- 
servation " — these  authors  add — "  renders  it  evident  that  the  agglu- 
tinating reaction  is  independent  of  the  state  of  immunisation." 
Analogous  cases  have  been  pointed  out  repeatedly  by  several 
observers. 

The  examples  cited  show,  on  the  one  hand,  that  the  serum  of 
individuals  endowed  with  acquired  immunity  may  be  without  any 
agglutinative  property,  but,  on  the  other,  that  this  power  may  be 
highly  developed  in  the  serum  of  susceptible  individuals.  The  argument 
based  on  these  data  may  be  corroborated  by  several  other  series  of 
facts.    Thus,  Salimbeni2  has  pointed  out  that  the  cholera  vibrio  is  not 
agglutinated  in  the  fluids  of  immunised  animals.    The  subcutaneous 
exudation  of  a  horse  treated  with  a  large  quantity  of  these  vibrios 
does  not  agglutinate  Koch's  vibrio  except  outside  the  body.    When 
this  exudation  is  drawn  off  shortly  after  the  injection  of  the  vibrios, 
the  organisms  render  the  fluid  uniformly  turbid.    But  a  short  ex- 
posure to  the  air  is  sufficient  to  bring  about  the  agglutination  of 
the   vibrios  in  the   same   exudation.     Guided  by  this  observation, 
Salimbeni  carried  out  comparative  experiments  on  the  action  of  the 
serum  of  vaccinated  animals  outside  the  body,  in  tubes  deprived 
of  oxygen  and  in  others  exposed  to  the  air.    In  the  former  agglu- 
tination did  not  take  place  or  was  very  incomplete,  in  the  latter  it  [275] 
soon  came  on.    This  fact  accords  perfectly  with  the  observation  of 
Pfeiffer's  phenomenon  in  the  peritoneal  cavity  of  guinea-pigs  from 
which  we  withdraw  a  fluid  containing  granules  that  have  resulted 
from  perfectly  isolated  vibrios.    In  other  micro-organisms  a  difference 
has  been  noted  in  this  respect.    Thus  Gheorghiewsky  has  seen  the 
agglutination  of  the  Bacillus  pyocyaneus  produced  under  the  in- 
fluence of  the  serum  of  vaccinated  animals,  even  in  tubes  deprived 
of  oxygen.    Durham  has  made  a  similar  observation  in  the  case  of 
the  typhoid  bacillus.    When,  however,  Trumpp3  wished  to  satisfy 
himself  as  to  the  agglutination  of  the  same  organism  in  the  body 
of  well-vacciuated  guinea-pigs,  he  obtained  only  imperfect  results. 
He  concluded  from  his  experiments  "that  the  formation  of  typhoid 
clumps   may  precede  the   breaking  down  of  the   bacteria  in  the 

1  Ann.  de  VInst.  Pasteur,  Paris,  1897,  t.  XI,  p.  411. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1897,  t.  xi,  p.  277. 

c  Arch.f.  Hyg.t  Miinchen  u.  Leipzig,  1898,  Bd.  xxxui,  S.  124. 


262  Chapter  IX 

animal  body  itself,  but  only  under  certain  conditions— when  the 
degree  of  immunity  of  the  animal  is  sufficiently  high  and  when 
the  bacilli  introduced  are  not  too  numerous  "  (p.  130).  In  the  case  of 
the  typhoid  bacillus,  a  certain  degree  of  agglutination  is  produced 
inside  the  animal  body,  but  it  is  markedly  increased  in  the  fluids 
that  have  been  withdrawn  and  exposed  to  the  action  of  the  air. 

It  has  been  demonstrated,  repeatedly,  that  the  agglutination  of 
micro-organisms  by  their  specific  serums  does  not  prevent  their 
growth  and  multiplication.  These  agglutinated  organisms  lose  none 
of  their  virulence.  Issaeff1,  working  in  my  laboratory,  carried  out 
an  investigation  on  this  point  in  the  case  of  the  pneumococcus. 
He  vaccinated  rabbits  against  this  organism  and  satisfied  himself 
that  the  organism  still  grows  well  in  the  blood  serum  of  such 
rabbits;  but,  instead  of  presenting  the  typical  form  of  lanceolate 
diplococci,  the  pneumococcus,  under  these  conditions,  forms  very 
long  chains  of  true  streptococci.  Having  filtered  the  cultures  in 
order  to  get  rid  of  the  serum,  he  injected  them  into  rabbits  and 
mice  and  demonstrated  that  the  pneumococci  had  retained  to  the 
full  their  initial  virulence.  Sanarelli2  carried  out  corresponding 
experiments  with  Gamaleia's  vibrio,  which,  as  we  know,  also  forms 
chains  in  the  serum  of  vaccinated  animals.  When  filtered  on  a  paper 
filter  and  washed  with  physiological  saline  solution,  the  vibrios  were 
found  to  be  just  as  virulent  as  were  the  control  vibrios  grown  in 
[276]  the  serum  of  susceptible  animals.  More  recently,  Mesnil3  demon- 
strated the  same  point  in  connection  with  the  bacillus  of  swine 
erysipelas.  He  experimented  on  cultures  that  were  agglutinated 
after  their  formation  and  also  on  others  agglutinated  as  they  were 
growing.  The  fluid  of  the  culture  was  decanted  and  replaced  by 
fresh  broth  until  the  elimination  of  the  serum  was  complete.  Mice, 
inoculated  with  the  washed  clumps,  died  in  the  normal  period,  thus 
affording  proof  that  "agglutination  in  no  way  alters  the  vitality 
and  virulence  of  the  bacillus  of  swine  erysipelas "  (p.  492). 

We  can  readily  understand,  after  the  demonstration  of  these 
various  facts,  that  it  is  impossible  to  maintain  Max  Gruber's  theory 
that  the  agglutinative  power  constitutes  the  fundamental  basis  of 
acquired  immunity.  Hence  this  writer,  after  publishing  several  pre- 
liminary notes  in  1896,  has  not  yet  decided  to  give  to  his  hypothesis 

1  Ann.  de  VInst.  Pasteur,  Paris,  1893,  t.  vn,  p.  260. 
a  Ann.  de  VInst.  Pasteur,  Paris,  1893,  t.  vn,  p  225. 
8  Ann.  de  VInst.  Pasteur,  Paris,  1898,  t  xn,  p.  481. 


Acquired  immunity  against  micro-organisms      263 

a  more  extended  development.    Nor  has  any  one  else  attempted  to 
defend  it. 

It  is  probable  that  in  certain  special  cases  the  immobilisation  of  very 
motile  bacteria  and  their  agglutination  into  clumps  may  facilitate  the 
reaction  of  the  animal  organism,  especially  the  rapidity  of  phagocy- 
tosis. Thus,  Besredka1  observed  that  guinea-pigs  when  inoculated 
with  typhoid  bacilli  that  had  previously  been  mixed  with  the  blood 
serum  of  normal  animals  survived.  The  most  active  amongst  these 
serums  was  ox  serum  heated  to  60°  C.  Guinea-pigs  furnished  a 
serum  which  was  much  less  active.  The  resistance  of  guinea-pigs, 
inoculated  into  the  peritoneal  cavity,  was  in  direct  ratio  to  the 
agglutinated  condition  of  the  bacilli.  Besredka  lays  stress  on  the 
facility  with  which  the  bacilli,  when  agglomerated  into  large  clumps, 
were  ingested  by  the  phagocytes,  and  suggests  that  there  is  a  certain 
stimulating  action  of  the  serums  on  the  leucocytes.  When  he  in- 
jected into  guinea-pigs  a  mixture  of  typhoid  bacilli  and  guinea-pig's 
serum,  made  immediately  before  injection,  his  animals  died  from 
infection.  But  when  he  left  the  bacilli  for  some  time  in  contact 
with  the  guinea-pig's  serum  outside  the  body,  and  did  not  inject 
the  mixture  until  after  agglutination  was  complete,  the  inoculated 
animals  usually  survived.  This  experiment  indicates  the  part  played 
by  agglutination  in  the  resistance  offered  by  the  animal,  and  at  the 
same  time  proves  that  in  the  body  of  the  guinea-pig  the  agglomera- 
tion of  the  micro-organisms  into  clumps  does  not  take  place  to  the  [277] 
same  degree  as  in  the  serum  prepared  in,  and  left  in  contact  with, 
the  air. 

In  any  case,  the  data  collected  by  Besredka  cannot  be  put 
forward  as  an  argument  in  favour  of  the  essential  part  played  by 
agglutination  in  acquired  immunity,  nor  can  they  weaken  the  facts 
indicated  as  to  the  absence  of  agglutinative  power  in  examples  of 
acquired  immunity  and  as  to  the  virulence  of  the  agglutinated  micro- 
organisms. The  part  played  by  agglutination  in  this  immunity  is 
merely  accidental  and  subordinate. 

Special  researches  have  been  carried  out  with  the  object  of  de- 
nning, exactly,  the  origin  of  agglutinins  in  the  body  of  an  animal 
that  has  acquired  immunity.  Observers  are  unanimous  in  recognising 
that,  of  all  parts  of  the  organism,  the  blood  is  richest  in  agglutinin. 
This  substance  is  found  in  the  blood  serum  as  well  as  in  the  plasma. 
From  this  (corroborated  by  the  agglutinative  property  of  other 
1  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  209. 


264  Chapter  IX 

fluids,  such  as  the  pericardial  fluid,  oedemas  very  poor  in  formed 
elements,  etc.)  it  follows  that  the  agglutinin  circulates  in  the 
blood  and  lymph  of  the  living  animal.  Several  observers,  amongst 
whom  I  may  cite  Achard  and  Bensaude1,  Arloing2,  and  Widal  and 
Sicard3,  put  to  themselves  the  question  whether,  before  passing 
into  the  blood,  the  agglutinin  is  not  formed  in  the  exudation  de- 
veloped at  the  seat  of  inoculation  of  the  micro-organisms.  Their 
conclusions  were  invariably  negative;  they  were  never  able  to  find 
more  agglutinins  in  these  exudations  than  in  .the  blood.  Pfeiffer 
and  Marx4  had  occasionally  observed  that  their  animals,  inoculated 
with  the  cholera  vibrio,  early  exhibited  an  agglutinative  power  in 
the  spleen;  but  this  result  was  not  met  with  sufficiently  constantly 
to  enable  them  to  draw  a  positive  conclusion.  A  little  later,  van 
Emden5  studied  in  detail  the  distribution  of  the  agglutinative  pro- 
perty in  the  body  of  an  animal  inoculated  with  Bacillus  aerogenes. 
His  researches  led  him  to  the  conclusion  that  the  spleen  and  the 
lymphoid  organs  must  be  regarded  as  the  source  of  the  agglutinins. 
[278]  Shortly  after  the  inoculation  of  the  bacilli,  an  extract  of  the  spleen 
wag  more  agglutinative  than  the  blood  or  any  of  the  other  organs. 
In  rabbits  from  which  the  spleen  had  been  removed,  the  same  rdle 
was  filled  by  the  bone  marrow  and  probably  also  by  the  lymphatic 
nodules.  But  this  preponderance  of  the  haematopoietic  organs  did 
not  continue  long,  the  blood  soon  becoming  the  most  important 
seat  of  the  agglutinative  power. 

The  proof  that  this  question  of  the  origin  of  the  agglutinins  is 
a  very  delicate  and  difficult  one  is  afforded  by  an  investigation  very 
carefully  carried  out  by  Gengou6  on  the  agglutination  of  the 
attenuated  anthrax  bacillus  (Pasteur's  first  vaccine)  by  the  fluids  and 
organs  of  normal  and  prepared  guinea-pigs.  This  observer  was  never 
able  to  obtain  any  confirmation  of  the  results  obtained  by  van  Emden 
with  another  micro-organism.  In  Gengou's  guinea-pigs  it  was  always 
the  blood  fluid  which  showed  itself  most  agglutinative,  the  organs 
exhibiting  merely  a  feeble  and  inconstant  agglutinative  power.  As  the 

1  Arch,  de  med.  exper.  et  d'anat.  path.,  Paris,  1896,  lre  serie,  t.  vnr,  p.  759.— 
Bensaude,  "Le  phenomene  de  1'aggluti nation  des  microbes,"  Paris,  1897,  p.  252. 

2  Compt.  rend.  Soc.  de  biol.,  Paris,  1897,  p.  104. 

1  Ann.  de  VInst.  Pasteur,  Paris,  1897,  t.  xi,  p.  376. 

4  Ztschr.f.  Hyg.,  Leipzig,  1898,  Bd.  xxvn,  S.  272. 

6  Ztschr.f.  Hyg.,  Leipzig,  1899,  Bd.  xxx,  S.  19. 

6  Arch,  internal,  de  Pharmacodyn.,  Gaud  et  Paris,  1899,  voL  vi,  p.  299. 


Acquired  immunity  against  micro-organisms      265 

extracts  of  leucocytes  were  always  found  to  be  markedly  less  active 
than  the  blood  and  the  fluids  of  the  exudations,  Gengou  was  obliged 
to  come  to  the  conclusion  that  the  agglutinins  cannot  be  regarded 
as  products  of  the  cells  of  the  animal  body;  this  he  sums  up  by 
saying  that  "in  the  increase  of  the  agglutinative  power  of  its  blood 
the  organism  of  the  animal  plays  only  a  relatively  passive  part" 

(p.  337). 

I  think  that,  in  spite  of  the  facts  established  by  Gengou,  his 
conclusion  can  scarcely  be  regarded  as  final.  The  agglutinative 
property,  developing  in  the  animal  body,  must  be  attributed  to 
some  cellular  influence,  because  we  know  that  the  prolonged  sojourn 
of  micro-organisms  in  the  animal  fluids  is  incapable  of  conferring 
on  them  this  power.  As  Gengou's  experiments  did  not  permit  him 
to  attribute  the  formation  of  agglutinin  to  any  formed  element,  it 
must  be  concluded  that,  although  perfectly  exact,  they  were  insuffi- 
cient to  solve  the  problem.  Geugou  killed  his  animals  at  a  stage  when 
their  blood  was  already  pretty  strongly  agglutinative.  At  this  stage 
the  organs  only  possessed  it  to  a  much  more  feeble  degree.  Perhaps, 
if  he  had  examined  his  animals  at  an  earlier  stage,  when  the  blood 
possessed  a  much  less  marked  agglutinative  power,  he  might  have 
obtained  a  more  powerful  agglutination  with  an  extract  of  the 
organs.  In  my  researches  on  the  resorption  of  cells,  I  observed,  on 
several  occasions,  that  the  abdominal  fluid  of  guinea-pigs  which  had  [279] 
received  an  injection  of  goose's  blood  became  agglutinative  before 
the  blood  serum.  Later,  however,  the  blood  exhibited  a  greater 
agglutinative  power  than  did  the  peritoneal  fluid.  If  to  this  fact 
we  add  the  results  of  van  Emden's  experiments,  we  shall  be  tempted 
to  assign  to  the  cells  found  in  the  peritoneal  exudation  and  in  the 
lymphoid  organs  a  share  in  the  production  of  the  agglutinin.  This 
question  of  the  origin  of  the  agglutinative  power  is,  however,  a  very 
difficult  one,  and  it  is  impossible,  in  the  imperfect  state  of  our 
knowledge,  to  express  oneself  in  a  more  positive  fashion.  Fortu- 
nately, according  to  the  whole  of  our  data  on  this  phenomenon, 
the  part  played  by  agglutination  in  immunity  can  only  be  very  incon- 
siderable, and  we  may  be  allowed  to  consider  our  general  problem 
without  concerning  ourselves  over  much  about  the  origin  of  the 
agglutinative  property. 

Among  the  definite  results  obtained  from  the  study  of  the  agglu- 
tinins, it  may  be  specially  pointed  out  that  these  substances  can 
in  no  way  be  identified  with  the  fixatives.  These  latter  were,  for 


266  Chapter  IX 

long,  spoken  of  as  preventive  substances.  They  are  so  termed  in 
the  early  papers  of  Jules  Bordet  treating  upon  this  question.  The 
explanation  of  this  designation  is  that,  for  a  series  of  years,  the 
presence  of  the  fixatives  was  revealed  chiefly  by  the  preventive  or 
protective  property  of  the  media  which  contained  them. 

To  gain  a  clear  conception  of  this  protective  property,  which 
occupies  so  important  a  place  in  the  study  of  acquired  immunity, 
we  must  go  back  to  an  epoch  in  our  science  when  it  was  sought 
to  prove  that  the  fluids  of  the  body  played  a  part  in  the  production 
of  immunity.  Shortly  after  the  earliest  researches  on  the  bacteri- 
cidal power  of  the  blood  had  been  made,  the  idea  of  applying  the 
results  obtained  in  this  direction  to  the  production  of  immunity  in 
animals  by  means  of  injections  of  blood  occurred.  The  first  step  in 
this  direction  was  taken  by  Richet  and  Hericourt1,  who  succeeded 
in  vaccinating  rabbits  against  a  variety  of  staphylococcus  by  means 
of  defibrinated  dog's  blood.  The  dog  is  naturally  refractory  against 
this  organism,  and  the  blood  of  a  normal  dog  exercised  a  certain 
vaccinal  or  protective  influence  on  rabbits  inoculated  with  the  staphy- 
lococcus. But  this  action  was  much  more  marked  when  Richet  and 
[280]  Hericourt  employed  the  defibrinated  blood  of  dogs  which  had  pre- 
viously received  inoculations  of  the  staphylococcus.  Shortly  after 
this  observation,  von  Behring2  made  his  discovery  of  antitoxins 
in  the  blood  serum  of  animals  immunised  against  tetanus  and 
diphtheria  toxins.  In  collaboration  with  Kitasato  he  demonstrated 
that  the  serum  of  these  animals,  when  injected  into  normal  animals, 
protected  them  against  intoxication  by  the  poisons  of  diphtheria 
and  tetanus.  This  great  discovery,  which  has  been  confirmed  on 
all  sides  and  extended  to  other  poisons,  gave  rise  to  the  view 
that  a  serum  exerting  any  protective  power  depends  solely  on  its 
property  of  impairing  the  action  of  the  toxins.  A  more  careful 
study  of  the  phenomena  which  appear  under  the  influence  of  the 
serums  has,  however,  demonstrated  the  inaccuracy  of  this  view. 
I  was  able  to  furnish  the  proof3  that  the  blood  serum  of  rabbits 
vaccinated  against  the  micro-organism  of  the  Gentilly  pneumo- 
enteritis  prevented  normal  rabbits  from  contracting  a  fatal  infection. 
Nevertheless,  the  serum  exerted  no  influence  on  the  toxin  of  this 

1  Compt.  rend.  Acad.  d.  Sc.,  Paris,  1888,  t.  cvn,  p.  750. 

2  Behring  u.  Kitasato,  Deutsche  med.  Wchnschr.,  Leipzig,  1890,  S.  1113. 

3  Ann.  de  VInst.  Pasteur,  Paris,  1892,  t.  vi,  p.  299. 


Acquired  immunity  against  micro-organisms      267 

micro-organism;  the  rabbits  that  received  the  minimal  lethal  dose 
of  this  toxin,  mixed  with  serum  from  vaccinated  rabbits,  died,  as 
did  the  control  animals,  from  rapid  poisoning.  It  was  evident  then 
that  this  serum,  which  prevented  infection  without  in  any  way 
hindering  intoxication,  could  not  be  classed  in  the  category  of  anti- 
toxic serums.  We  find  ourselves,  therefore,  in  the  presence  of 
a  new  property  of  the  fluids  of  the  body  to  which  we  have  given 
the  name  of  protective  or  anti-infective  power.  We  are  driven  to 
this  conclusion  the  more  as  the  serum  in  question  was  neither 
bactericidal  nor  agglutinative. 

This  discovery  was  soon  confirmed  by  R.  Pfeiffer1  for  the  cholera 
vibrio.  Animals  vaccinated  against  this  organism  furnished  Pfeiffer 
with  a  serum  which,  whilst  not  at  all  antitoxic,  was  distinctly  pro- 
tective when  injected  into  normal  guinea-pigs.  It  protected  these 
animals  from  a  fatal  infection  by  the  vibrio  and,  when  injected  into 
the  peritoneal  cavity,  it  set  up  the  granular  transformation  of  the 
cholera  vibrios, — Pfeiffer's  phenomenon.  Pfeiffer,  for  this  reason, 
gave  to  the  protective  anti-vibrio  serum  the  name  of  bactericidal 
serum.  As  the  granular  transformation  was  produced,  under  the  [281] 
influence  of  this  serum,  with  cholera  vibrios  only  and  never  with 
other  species  of  vibrio,  Pfeiffer  gave  to  the  active  substance  in 
the  serum  the  name  of  specific  cholera-antibody.  This  substance, 
according  to  his  theory,  was  formed  in  the  animal  body  at  the 
expense  of  an  inactive  antibody  which  became  transformed  into  an 
active  substance  under  the  influence  of  the  peritoneal  endothelium. 

The  possibility  of  thus  vaccinating  susceptible  animals  by  means 
of  the  serums  of  immunised  animals,  quite  apart  from  any  anti- 
toxic power,  was  easily  confirmed  and  extended  to  several  other 
infective  diseases.  Pfeiffer  and  Kolle2,  Funck3,  Chantemesse  and 
Widal4  demonstrated  it  in  connection  with  the  experimental  disease 
produced  in  animals  by  the  typhoid  bacillus;  Loeffler  and  Abel5  for 
the  Bacillus  coli,  etc.  The  protective  or  anti-infective  power  of  the 
serum  and  other  fluids  of  immunised  animals  was  soon  recognised 
as  a  general  property. 

1  Zischr.f.  Hyg^  Leipzig,  1894,  Bd.  xvi,  S.  268;  1894,  Bd.  xvnr,  S.  1. 
-  Ztschr.  f.  Hyg.,  Leipzig,  1896,  Bd.  xxi,  S.  203 ;  Deutsche  med.   Wchnschr^ 
Leipzig,  1896,  SS.  185,  735. 

3  "  La  serotherapie  de  la  fievre  typhoide,"  Bruxelles,  1896. 

4  Bull.  Soc.  med.  d.  hop.,  Paris,  1893,  27  Janvier. 

5  Centralbl  f.  Bakteriol.  u.  Parasitenk..  Jena,  1896,  Ite  Abt.,  Bd.  xix,  S.  51 ; 
Festschr.  z.  lOOjdhr.  Stiftungsfeier  d.  med.  chir.  Friedr.  Wilh.  Instituts,  1895. 


268  Chapter  IX 

Pfeiffer  and  his  collaborators,  as  well  as  many  other  investi- 
gators, laid  special  stress  on  the  bactericidal  character  of  these 
protective  fluids.  It  was  seen  that  the  serums  of  immunised  ani- 
mals were  often  almost  or  completely  incapable  of  killing  the 
corresponding  micro-organisms,  but  they  were  still  regarded  as 
bactericidal,  because,  when  injected  into  the  peritoneal  cavity  of 
normal  animals,  they  set  up  the  transformation  of  vibrios  into 
granules,  or,  in  the  case  of  other  bacteria,  determined  certain  phe- 
nomena of  extracellular  destruction.  Whilst  carrying  on  researches 
in  this  direction,  Frankel  and  Sobernheim1  discovered  a  fact  of  great 
importance.  They  found  that  the  protective  substance  of  the  serum 
of  animals  vaccinated  against  the  vibrios  resisted  heating  to  70°  C. 
When  submitted  to  the  influence  of  this  temperature,  the  serum  lost 
its  bactericidal  power  completely,  but  remained  quite  as  protective 
as  the  unheated  serum,  when  injected  into  susceptible  animals.  This 
experiment,  which  has  since  been  confirmed  repeatedly,  furnished 
us  with  a  means  of  separating  the  bactericidal  power  from  the 
[282]  protective  power  in  cases  where  both  were  present  in  the  same 
serum.  Later,  in  the  hands  of  Bordet,  it  proved  to  be  of  great 
service  in  connection  with  his  researches  on  the  concurrence  of  two 
substances  in  acquired  immunity. 

The  possibility  of  obtaining  Pfeifler's  phenomenon  outside  the  body 
by  "reactivating"  the  protective  serum  with  peritoneal  fluid  or  blood 
serum  of  normal  unvaccinated  animals  has  still  further  facilitated 
the  study  of  the  action  of  the  two  substances  in  acquired  immunity. 
It  was  with  the  help  of  this  method  that  Bordet  was  able  to  furnish 
so  much  valuable  information  on  the  subject  of  anti-cholera  serums 
and,  later,  on  that  of  haemolytic  serums.  The  discovery  by  Ehrlich 
and  Morgenroth2  of  the  fixation  by  the  sensitive  elements  of  the 
heat-resisting  (thermostabile)  substance  (that  which  resists  a  tem- 
perature of  65° — 70°  C.)  constitutes  a  new  and  important  acquisition 
to  the  study  of  acquired  immunity.  The  discovery  has  been  applied 
by  Bordet  to  micro-organisms,  and  since  then  it  has  been  found 
possible  to  study  much  more  precisely  the  mode  of  action  of 
specific  protective  serums. 

Even  before  this  last  scientific  advance  had  been  made  it  was  pos- 
sible to  determine  the  relations  between  the  protective  power  and  the 
agglutinative  power  of  the  fluids  of  animals  that  had  acquired 

1  Hygien.  Rundschau,  Berlin,  1894,  iv  Jahrg.,  SS.  97,  145. 

2  Berl  klin.  Wchnschr.,  1899,  S.  6. 


Acquired  immunity  against  micro-organisms      269 

immunity.  Both  resist  about  the  same  temperatures;  both  are 
found  in  the  blood  plasma  and  pass  into  the  fluids  of  exudations 
and  tran sudations.  But  it  may  be  affirmed  with  certainty,  as  already 
stated,  that  the  two  properties  are  quite  distinct.  Pfeiffer  has  laid 
great  stress  on  the  fact  that  highly  protective  serums  often  exhibit 
only  a  feeble  agglutinative  power  and  vice  versa.  During  an  in- 
vestigation1 into  an  epidemic  of  typhoid  fever,  he  had  occasion  to 
study  the  serum  of  patients  convalescent  from  this  disease.  The 
exact  dosage  of  the  two  properties  demonstrated  that  a  slightly 
marked  agglutinative  property  might  be  associated  with  a  very 
powerful  protective  property.  Gheorghiewsky2  made  similar  obser- 
vations on  animals  vaccinated  against  the  Bacillus  pyocyaneus. 
The  serum  of  a  goat,  although  more  agglutinative,  invariably  proved 
to  be  less  protective  than  that  of  a  rabbit.  A  similar  result 
was  obtained  with  the  serum  of  immunised  guinea-pigs.  "This [283] 
shows  distinctly  " — concludes  Gheorghiewsky — "  that  the  property 
possessed  by  serums  of  agglutinating  the  Bacillus  pyocyaneus  does 
not  march  parallel  with  the  protective  property  "  (p.  304).  Analogous 
examples  are  sufficiently  numerous  to  justify  us  in  accepting  the 
distinctiveness  of  the  two  properties  of  specific  serums. 

The  protective  or  anti-infective  substance  is,  therefore,  not  the 
same  as  the  agglutinin.  But  are  we  justified  in  regarding  it  as 
identical  with  the  fixative  substance,  or  fixative  (sensibilising  sub- 
stance, immunising  or  intermediary  substance,  or  amboceptor)  ?  From 
the  fact  that  the  fixative  was  at  first  rightly  designated  by  Bordet 
as  protective  substance  we  should  conclude  in  the  affirmative.  The 
question  is  an  important  one  and  merits  close  examination.  The 
discovery  of  an  exact  method  of  determining  the  presence  of 
fixatives  has  rendered  it  possible  to  ascertain  whether  these  sub- 
stances are  always  found  in  the  protective  fluids  and  also  whether 
the  presence  of  fixatives  necessarily  implies  the  protective  power 
of  the  serums. 

The  first  of  these  questions  has  been  answered  in  the  affirmative. 
All  the  protective  serums  studied  from  this  point  of  view,  by  Bordet 
and  Gengou>  were  found  to  be  endowed  with  very  distinct  fixative 
properties.  They  also  found  the  specific  fixative  in  the  serum  of 
guinea-pigs  immunised  with  the  attenuated  bacilli  of  the  first  vaccine 
of  Pasteur.  Now  this  serum  is  powerless  to  prevent  the  production 

1  "  Typhusepidemien  und  Trinkwasser,"  Jena,  1898,  S.  26. 

2  Ann.  de  I'Inst.  Pasteur,  Paris,  1899,  t.  xin,  p.  298. 


270  Chapter  IX 

of  fatal  infection  in  mice  into  which  is  simultaneously  injected  the 
bacillus  of  the  first  vaccine.  Consequently  a  fixative  fluid  is  not 
necessarily  protective.  This  is  in  accordance  with  the  fact  that  the 
micro-organisms  that  have  absorbed  the  fixative  may,  nevertheless, 
retain  their  virulence.  We  have  already  cited  the  experiment  of 
Mesnil  that  the  bacilli  of  swine  erysipelas,  mixed  with  the  specific 
serum  and  then  deprived  of  this  fluid,  produce  a  fatal  infection  in 
mice.  We  have  also  drawn  attention  to  the  fact,  demonstrated  by 
Sawtchenko,  that  anthrax  bacilli,  obtained  from  the  exudation  of 
immunised  rats,  give  rise  to  a  fatal  anthrax  in  normal  guinea-pigs 
and  rats.  The  experiments  of  Bordet  and  Gengou  proved  that  there 
is  absorption  of  the  fixative  substance  by  the  bacilli  of  swine 
erysipelas  and  of  anthrax  when  placed  in  contact  with  the  specific 
serums  of  the  immunised  animals.  In  order  that  the  protective 
power  may  manifest  itself  adequately,  therefore,  besides  the  fixative 
substance,  some  other  factor  capable  of  acting  is  also  necessary. 
[284]  In  connection  with  my  work  on  immunity  against  the  micro- 
organism of  swine  pneumo-enteritis  I  was  able  to  demonstrate  that 
the  serum  of  vaccinated  rabbits,  incapable  of  preventing  the  multi- 
plication of  the  specific  cocco-bacillus,  is  also  powerless  to  deprive  it 
of  its  virulence ;  it  is  without  the  power  of  causing  its  agglutination 
or  of  neutralising  its  toxin.  In  short,  this  serum  appears  to  exercise 
no  direct  action  on  the  micro-organism,  yet,  in  spite  of  that,  it 
prevents  its  pathogenic  action.  With  these  results  before  me, 
I  was  led  to  assume  a  certain  stimulating  action  of  the  serum  on 
the  defensive  elements  of  the  animal  organism  and  especially  on  the 
phagocytic  system.  The  discovery  of  the  fixative  property  of  serums 
would  lead  us  to  believe  that  this  stimulation  was  entirely  useless, 
and  that  the  permeation  of  micro-organisms  by  the  fixative  was 
amply  sufficient  to  bring  about  their  destruction  and  removal  from 
the  animal.  A  living  micro-organism  in  its  normal  form,  endowed  with 
full  virulence  and  provided  with  its  fighting  weapon,  the  toxin,  but 
at  the  same  time  permeated  by  the  fixative  substance,  might  behave 
in  the  animal  in  some  special  way.  It  might  excite  a  strong  posi- 
tive chemiotaxis  of  the  leucocytes  and  be  ingested  and  destroyed  by 
these  cells  with  greater  facility.  A  priori,  there  would  be  nothing 
to  object  to  in  this  view,  but  certain  facts  are  opposed  to  it.  Thus, 
in  the  case  of  micro-organisms  just  cited,  we  see  bacteria,  permeated 
not  only  with  the  fixative  but  also  with  cytases,  capable  of  producing 
a  fatal  infection.  We  are  thus  compelled  to  accept  the  theory  of  an 


Acquired  immunity  against  micro-organisms       271 

influence  of  protective  serums  not  only  on  the  micro-organisms  but 
also  on  the  organism  of  the  animal  into  which  they  are  introduced. 
As  this  influence  manifests  itself  in  the  form  of  a  strong  phagocytosis, 
it  is  only  natural  that  we  should  attribute  it  to  the  existence  of 
a  stimulating  action  of  the  serums  of  vaccinated  animals  on  the 
phagocytes  of  the  normal  animals.  The  detailed  analysis  of  the 
mechanism  of  the  immunity  acquired  as  the  result  of  the  injection 
of  these  serums,  as  we  shall  attempt  to  prove  in  the  following 
chapter,  in  many  cases  confirms  this  view. 

The  important  part  played  by  the  stimulation  of  the  phagocytic 
reaction  in  acquired  immunity  is  supported  by  yet  another  series  of 
facts  and  from  a  different  side.  It  has  been  clearly  established  that 
not  only  the  serum  of  immunised  animals  but  also  that  of  normal 
man  and  normal  animals,  themselves  susceptible  to  the  pathogenic 
action  of  the  micro-organisms,  protects  the  animal  organism  against 
infection.  This  fact  was  first  demonstrated  in  connection  with  re- 
searches on  the  vaccination  of  guinea-pigs  against  the  experimental  [285] 
peritonitis  produced  by  the  cholera  vibrio. 

G.  Klemperer1  was  the  first  to  observe  that  the  blood  of  several 
individuals  who  had  never  had  cholera  was,  nevertheless,  in  the 
case  of  guinea-pigs,  protective  against  peritoneal  infection  by  the 
cholera  vibrio.  He  concluded  therefrom  that  the  individuals  who 
had  furnished  this  protective  blood  possessed  immunity  against 
cholera.  Soon  afterwards  I2  was  able  to  extend  analogous  re- 
searches over  a  large  number  of  persons  and  to  show  that  the 
protective  power  of  the  blood  is  of  very  wide  distribution  in  human 
beings.  But,  instead  of  assuming  that  all  these  individuals,  whose 
fluids  protect  the  guinea-pig  from  peritoneal  infection,  possess  a 
natural  immunity  against  cholera,  I  came  to  the  conclusion  that 
the  protective  power  of  the  blood  cannot  be  taken  as  a  measure 
of  the  immunity  of  the  individual  from  whom  the  blood  was  drawn. 
Here  again  I  assumed  a  stimulant  action  of  the  human  blood  on 
the  phagocytic  reaction  of  the  guinea-pig,  looking  upon  it  as  quite 
natural  that  the  blood,  capable  of  exciting  the  reaction  in  an  alien 
animal,  might  remain  inactive  in  the  body  of  the  animal  which 
furnished  it. 

R.  Pfeiffer3  has  given  much  attention  to  the  protective  action 

1  Berl.  Jdin.  Wchnschr.,  1892,  S.  970. 

2  Ann.  de  Clnst.  Pasteur,  Paris,  1893,  t.  vn,  p.  411. 

3  Ztschr.f.  Hyg.,  Leipzig,  1894,  Bd.  xvi,  S.  268. 


272  Chapter  IX 

of  serums;  he  has  laid  special  stress  on  the  essential  difference 
between  the  influence  of  normal  serums  and  of  those  obtained  from 
animals  that  have  acquired  immunity.  Whilst,  in  order  to  obtain 
a  protective  effect  with  the  normal  blood  or  serum  of  man  and 
animals,  it  is  necessary  to  inject  a  considerable  quantity  (from 
0'5  c.c.  upwards),  the  specific  serum,  i.e.  serum  obtained  from 
persons  recovered  from  cholera  or  from  animals  vaccinated  against 
the  cholera  vibrio,  is  active  in  a  very  minute  dose.  Sometimes  the 
cholera  peritonitis  of  the  guinea-pig  is  prevented  by  a  fraction  of 
a  milligramme  of  such  serum1.  Based  on  these  facts,  Pfeiffer  has 
expressed  the  view  that  the  normal  serum  acts  by  stimulating 
the  natural  powers  of  defence  of  the  animal,  whilst  the  specific 
serum  exercises  its  influence  in  virtue  of  the  property  of  causing 
the  formation  of  a  special  secretion  which  acts  only  against  the 
micro-organism  which  served  for  the  production  of  the  immunity. 
Pfeiffer  and  his  collaborators  have  demonstrated  that  normal  serums 
are  protective,  not  only  against  the  cholera  vibrio,  but  also  against 
several  other  micro-organisms,  e.g.  the  typhoid  bacillus.  One  of  his 
[286]  pupils,  Voges2,  believed  that,  in  certain  infections,  the  protective 
power  of  normal  blood  might  be  greatly  exaggerated,  and  that, 
in  these  cases,  the  limit  between  the  activity  of  normal  and  of 
specific  serums  might  be  almost  completely  effaced.  He  affirmed, 
especially,  that  very  small  doses  (0*1  c.c.)  of  blood  serum  from 
a  normal  guinea-pig  was  quite  sufficient  to  prevent,  in  other  guinea- 
pigs,  a  fatal  infection  by  the  micro-organism  of  hog  cholera  and 
its  allies.  As  this  fact  might  be  of  general  application  I  asked 
M.  Saltykoff3,  who  was  working  in  my  laboratory,  to  verify  the 
statements  of  Voges.  Several  series  of  experiments  demonstrated 
the  incorrectness  of  the  contention.  The  small  doses  of  normal 
serum  of  guinea-pigs,  indicated  by  Voges,  were  found  to  be  ab- 
solutely incapable  of  protecting  against  the  virus  used  by  him  in 
his  experiments. 

The  fact  that  normal  serums,  injected  in  sufficiently  large  doses, 
exhibited  an  undoubted  protective  property,  affords  additional 
proof  that  this  property  cannot  be  identified  with  the  fixative 
power.  The  latter  was  present  in  serums  which  were  not  pro- 
tective ;  here,  then,  we  have  the  inverse  phenomenon  and  we  see 

1  See  Lazarus,  Berl  klin.  Wchnschr.,  1892,  S.  1072. 

2  Ztschr.f.  Hyg.,  Leipzig,  1896,  Bd.  xxin,  S.  149. 

3  Ann.  de  VInst.  Pasteur,  Paris,  1902,  t.  xvi,  p.  94. 


Acquired  immunity  against  micro-organisms       273 

normal  serums  exercise  their  protective  action  although  they  contain 
no  fixative.  This  follows  from  Bordet  and  Gengou's  experiments 
already  described,  according  to  which  the  cytases,  placed  in  contact 
with  micro-organisms  in  normal  serums,  remain  free,  simply  because 
of  the  absence  of  fixatives. 

We  are  led,  then,  from  these  demonstrations  to  recognise  the 
presence  of  stimulins  not  only  in  specific  serums,  but  also  in  normal 
serums.  Between  the  two  there  is  this  difference  that,  when  applied 
with  the  normal  fluids,  the  stimulins  alone  act,  whilst  when  injected 
with  the  serum  of  the  animal  enjoying  acquired  immunity  the  action 
of  the  stimulins  is  facilitated  and  reinforced  by  the  fixatives  or 
sometimes,  perhaps,  by  the  agglutinins. 

The  stimulating  influence  of  certain  normal  serums  may  be  so 
considerable  that  it  may  prevent  infection  by  the  micro-organism, 
injected  at  the  same  time  in  a  dose  many  times  more  than  lethal. 
Wassermann1  protected  guinea-pigs  by  injecting  into  the  peritoneal  [287] 
cavity  a  quantity  as  great  as  40  times  the  lethal  dose  of  typhoid 
bacilli,  by  introducing  at  the  same  time  and  at  the  same  place  3  c.c. 
of  normal  rabbit's  serum,  heated  to  60°  C.  Besredka2,  who  confirmed 
this  observation,  has  analysed  its  special  mechanism.  He  showed 
that  the  serum  exercises  a  very  marked  stimulating  influence  on 
the  guinea-pig's  leucocytes,  which  then  exhibit  a  truly  extraordinary 
phagocytic  activity.  They  are  seen  to  act  in  the  peritoneal  fluid, 
but  they  are  much  more  active  in  the  region  of  the  omentum,  where 
the  leucocytes  gorge  themselves  with  micro-organisms,  devouring 
them  by  dozens.  The  stimulating  action  of  the  heated  rabbit's  serum 
is  exercised  in  a  similar  fashion  if,  instead  of  micro-organisms,  grains 
of  carmine  be  injected.  Very  shortly  after  the  commencement  of 
the  experiment  very  little  carmine  is  found  outside  the  cells ;  it  is 
all  either  ingested  by  individual  leucocytes,  if  the  grains  are  small, 
or  surrounded  by  numerous  leucocytes  when  the  grains  are  massed 
together  ;  this  phagocytosis  is  most  developed  in  the  region  of  the 
omentum,  exactly  as  in  the  case  of  typhoid  bacilli. 

These  facts,  which  so  clearly  demonstrate  the  stimulating  action 
of  the  normal  rabbit's  serum,  prove  in  another  way  that  the  stimulin 
resists  heating  to  60°  C.,  and  that,  in  this  respect,  it  resembles  the 
agglutinins  and  fixatives.  This  may  afford  us  an  indication  as  to  the 
nature  of  the  stimulating  substance,  The  possibility  of  obtaining  an 

1  Deutsche  med.  Wchnschr.,  Leipzig,  1901,  S.  4. 

2  Ann.  de  TInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  209. 

B.  18 


274  Chapter  IX 

antistimulin  gives  us  another  valuable  indicatiou.  Wassermann, 
in  the  work  we  have  just  cited,  showed  that  the  serum  of  a  rabbit, 
previously  treated  with  guinea-pig's  serum  and  injected  under  the 
same  conditions  as  in  the  experiment  with  normal  rabbit's  serum,  has 
completely  lost  its  protective  power.  The  typhoid  bacilli  multiply 
freely  in  the  peritoneal  cavity  and  the  organism  of  the  guinea-pig 
is  incapable  of  opposing  a  sufficient  resistance.  Wassermaun  thinks 
that,  in  this  case,  the  disease  becomes  grave  because  of  the  anticytase 
found  in  the  serum  of  rabbits  treated  with  guinea-pig's  blood.  There 
is  no  doubt  that  this  serum  is  really  anticytasic.  But  as  the  free 
cytases  found  in  the  peritoneal  cavity  of  a  guinea-pig  inoculated  at 
the  moment  of  phagolysis,  become  inactive  under  the  influence  of 
the  anticytase  and  play  merely  a  minor  part,  it  is  impossible  to  ac- 
[288]  cept  the  German  investigator's  interpretation.  Indeed,  Besredka  has 
proved  that,  in  this  case,  it  is  the  antiphagocytic  or  antistimulant 
action  of  the  rabbit's  serum  which  brings  about  the  fatal  issue  in 
the  case  of  the  typhoid  inoculation. 

We  have  laid  stress  on  the  point  that  an  animal,  whose  serum  is 
protective  when  introduced  into  another  animal,  may  itself  not  be 
refractory  against  the  specific  micro-organism.  As  regards  the  serum 
of  normal  unvaccinated  animals  this  has  been  so  fully  demonstrated 
that  nowadays  no  one  doubts  it.  The  question  is  more  complicated 
in  the  case  of  animals  that  have  acquired  immunity.  As  in  the 
great  majority  of  cases  the  serum  of  these  animals  is  found  to  be 
endowed  with  a  very  great  protective  power,  it  has  been  accepted 
as  proved  that  the  animal  which  furnishes  it  must  itself  possess 
great  immunity.  The  degree  of  protective  power  has  even  been  taken 
as  the  measure  of  the  acquired  immunity.  Thus,  the  numerous 
attempts  to  vaccinate  the  human  subject  against  typhoid  fever, 
undertaken  in  consequence  of  the  researches  of  PfeifFer  and  Kolle1, 
were  based  on  the  fact  that  in  these  cases  the  serum  of  vaccinated 
individuals  acquires  a  great  protective  power.  It  was  argued  that 
if  this  power  is  present  it  can  only  be  due  to  the  acquired  immunity 
of  the  individuals  who  furnish  such  a  serum.  Undoubtedly  the  pro- 
tective property  of  the  fluids  and  the  resistance  are  often  equal ;  but 
it  is  none  the  less  true  that  there  are  cases  where;  in  spite  of  this 
property  being  markedly  developed,  the  animal  that  furnishes  the 
protective  serum  is  susceptible  to  the  action  of  the  micro-organism 
and  may  even  succumb  to  infection  therewith. 

1  Ztschr.f.  Hyg.,  Leipzig,  1896,  Bd.  xxi,  S.  203. 


Acquired  immunity  against  micro-organisms      275 

As  the  hypothesis  just  mentioned  is  of  importance  from  a 
general  point  of  view  it  must  be  supported  by  adequate  proof.  It 
was  during  the  course  of  the  vaccination  of  rabbits  against  the 
micro-organism  of  the  pneumo-enteritis  epidemic  at  Gentilly  that 
I  was  first  able1  to  assure  myself  of  its  accuracy.  I  noticed  that 
some  of  these  rabbits,  although  vaccinated,  ultimately  succumbed 
to  pyaemia,  set  up  solely  by  this  micro-organism.  They  were  con- 
sequently not  refractory  against  the  disease,  and  yet  their  blood 
serum,  when  injected  into  normal  rabbits  along  with  an  absolutely 
fatal  dose  of  micro-organisms,  was  found  to  be  highly  protective. 
This  observation  drove  me  to  the  conclusion  that  the  protective 
power  is  not  a  function  of  immunity  and  cannot  be  received  as  a 
measure  of  this  immunity.  Analogous  facts  have  since  been  demon- 
strated in  certain  other  cases.  Thus,  Pfeiffer2  on  several  occasions  has  [289] 
found  that  guinea-pigs,  highly  immunised  against  the  cholera  vibrio, 
have  succumbed  after  the  injection  of  a  moderate  quantity  of  these 
organisms.  "On  post-mortem  examination  of  these  cases  living 
vibrios  were  found  in  the  peritoneal  cavity,  sometimes  in  considerable 
numbers  ;  and  yet  minimal  doses  of  the  heart  blood  given  to  normal 
guinea-pigs  caused  in  these  animals  a  very  marked  breaking  down  of 
the  vibrios."  Alongside  these  facts  may  be  placed  others,  described 
in  the  preceding  chapter,  of  well-immunised  animals  dying  from  infec- 
tion, after  they  had  been  weakened  by  opium,  cold,  or  other  lowering 
agent.  It  is  clearly  seen,  then,  that  for  the  manifestation  of  acquired 
immunity  it  is  necessary  that  the  reaction  of  the  living  cell  elements 
should  take  place  without  let  or  hindrance.  When  this  reaction  fails, 
the  possession  of  even  great  protective  power  is  insufficient  to  prevent 
the  immunised  animal  from  contracting  a  fatal  infection. 

If,  in  acquired  immunity  against  micro-organisms,  it  is  really  the 
cell  defence  which  plays  the  most  important  part,  we  can  readily 
imagine  cases  where  it  by  itself  can  confer  immunity  without  calling 
in  the  co-operation  of  the  protective  power  of  the  fluids.  When  in 
this  connection  we  study  the  resistance  of  an  animal  against  various 
pathogenic  organisms,  we  note,  first  of  all,  the  very  great  variability 
that  exists  in  the  production  of  the  acquired  humoral  properties.  In 
certain  cases,  as  in  vaccination  against  vibrios  or  typhoid  bacilli,  the 
serum  very  readily  becomes  not  only  protective,  but  agglutinative 
and  fixative.  In  other  cases  these  properties  develop  with  difficulty 


1  Ann.  de  VInst.  Pasteur,  Paris,  1892,  t.  vi,  p.  300. 
1  Ztschr.f.  Hyg.,  Leipzig,  1895,  Bd.  xix,  S.  82, 


18—2 


276  Chapter  IX 

and  are  only  manifested  after  a  long  period  of  vaccination.  Such  is 
the  case  with  anthrax.  After  the  discovery  of  protective  serums, 
numerous  attempts  were  made  to  obtain  a  serum  protective  against 
the  anthrax  bacillus.  Several  observers  failed  in  their  attempts, 
others  were  more  fortunate.  Sclavo1  and  Marchoux2  were  the  first 
to  succeed  in  obtaining  a  protective  serum  from  animals  hyper- 
immunised  against  anthrax.  They  were  able  to  show  that  the 
serum  of  sheep,  treated  first  with  vaccines  and  then  repeatedly 
with  anthrax  virus,  would  protect  rabbits  against  a  fatal  dose 
[290]  of  the  bacillus.  Marchoux  even  obtained,  with  hyperimmunised 
rabbits,  a  serum  which  prevented  normal  rabbits  from  contract- 
ing fatal  anthrax.  Sobernheim3  was  less  fortunate  in  his  first 
experiments.  He  satisfied  himself  that  the  blood  serum  of  cattle 
that  had  recovered  spontaneously  from  anthrax  or  that  had  been 
vaccinated  according  to  Pasteur's  method,  was  absolutely  unable  to 
protect  small  animals  against  the  anthrax  bacillus,  and  his  hyper- 
vaccinated  rabbits  furnished  serums  of  doubtful  activity.  It  was 
only  later  that  he  succeeded4  in  obtaining  better  results  ;  especially 
when  he  used  sheep.  Even  then  he  found  that  in  the  production 
of  the  anti-infective  property  the  individuality  of  the  immunised 
animals  had  a  dominant  influence.  Thus,  in  two  sheep,  treated  in 
exactly  the  same  way,  the  serum  of  one  was  found  to  be  incapable  of 
protecting  a  rabbit,  whilst  that  of  the  other  exhibited  an  undoubted, 
although  feeble,  protective  power. 

But  what  is  of  greater  interest  to  us,  from  our  point  of  view,  is 
that  guinea-pigs  which  have  been  vaccinated  against  anthrax  and 
which  enjoy  a  considerable  immunity  against  this  disease,  exhibit  no 
protective  power.  In  a  letter  from  Behring  I  learnt  that  this  fact 
had  for  the  first  time  been  demonstrated  by  Wernicke  in  experiments 
carried  out  in  the  Hygienic  Institute  at  Marburg.  After  repeated 
and  painstaking  attempts  this  observer  succeeded  in  vaccinating 
guinea-pigs  against  enormous  doses  of  virulent  anthrax  bacilli.  The 
serum  from  the  animals  so  immunised  was,  however,  quite  incapable 
of  protecting  normal  guinea-pigs  against  a  fatal  infection.  This  result 
was  the  more  extraordinary  since  Wernicke's  pigeons,  likewise  vac- 

1  \CentralU.f.  Bakteriol.  u.  Parasitenk.,  Jena,  1895,  Bd.  xvm,  S.  744];  Riv» 
(fig.  e  San.  Ptibbl.,  Torino,  1896,  t.  vn,  nos.  18—19 ;  ibid.  1901,  t.  xn,  p.  212. 

2  Ann.  de  Vlnst.  Pasteur,  Paris,  1895,  t.  ix,  p.  785. 

3  Ztschr.f.  Hyg.,  Leipzig,  1897,  Bd.  xxv,  S.  301. 

4  Ztschr.  f.  Hyg.,  Leipzig,  1899,  Bd.  xxxi,  S.  89. 


Acquired  immunity  against  micro-organisms      277 

cinated  against  anthrax,  gave  a  serum  whose  protective  power  was 
quite  distinct.  Realising  the  great  importance  of  these  facts  I  asked 
M.  de  Xittis1  to  repeat  these  experiments  in  my  laboratory.  The 
vaccination  of  pigeons  is  an  easy  matter,  but  that  of  guinea-pigs 
presents  great  difficulties.  He  succeeded,  nevertheless,  in  vaccinating 
some  of  these  rodents  very  highly,  and  this  enabled  him  to  compare 
the  protective  power  of  the  blood  serum  in  the  two  species.  That  of 
the  vaccinated  pigeon  was  found  to  be  endowed  with  this  power  and 
protected  guinea-pigs  and  mice  against  virulent  anthrax.  The  serum 
of  the  immunised  guinea-pigs,  on  the  contrary,  exhibited  no  pro-[29i] 
tective  property,  just  as  in  Wernicke's  experiments.  The  guinea-pigs 
and  mice,  into  which  this  serum  was  injected  at  the  same  time  as 
the  anthrax  bacilli,  died  even  when  attenuated  anthrax  was  used. 
We  have,  then,  in  this  case,  an  example  of  acquired  immunity,  inde- 
pendent of  any  protective  power  of  the  fluids  of  the  body. 

In  the  course  of  their  researches  on  the  bacillus  isolated  by 
R.  Pfeiffer  from  persons  attacked  by  influenza,  Delius  and  Kolle2 
tried  to  vaccinate  susceptible  animals  (guinea-pigs)  against  this 
minute  organism  and  to  immunise  animals  naturally  refractory  (dog, 
sheep,  goat)  against  fairly  large  doses  of  cultures.  They  succeeded 
in  vaccinating  guinea-pigs  against  ten  times  the  lethal  dose,  but 
never  obtained  any  protective  serum.  Nor  did  the  other  animals 
that  were  treated  furnish  a  protective  serum.  "From  the  whole  of 
our  experiments  carried  on  for  several  years  " — conclude  Delius  and 
Kolle — "it  is  quite  evident  that  we  were  unable  to  produce  any 
appreciable  change  in  the  blood  by  the  use  of  those  methods  which 
have  produced  specific  immunising  serums  against  other  bacteria 
such  as  the  bacilli  of  diphtheria,  cholera,  typhoid  fever,  and  'blue 
pus'"  (p.  345).  Slatineano  undertook  a  detailed  study  of  Pfeifier's 
bacillus  in  my  laboratory,  but  he  found  it  impossible  to  demonstrate 
any  unquestionable  protective  effect  exerted  by  the  blood  serum  of 
vaccinated  guinea-pigs  upon  normal  guinea-pigs  inoculated  with  a 
fatal  dose  of  this  organism.  We  are  not  justified,  therefore,  in  classing 
this  bacillus  with  the  anthrax  bacillus  ;  we  may,  however,  cite  it  as 
an  argument  illustrating  the  difficulty  that  is  met  with,  in  certain 
examples  of  acquired  immunity,  of  discovering  the  protective  power, 
when  feeble  and  masked. 

The  inoculation  with  micro-organisms  of  animal  nature  causes  the 

1  Ann.  de  llnst.  Pasteur,  Paris,  1901,  t.  xv,  p.  769. 

2  Ztschr.f.  Hyg.,  Leipzig,  1897,  Bd.  xxiv  S.  327. 


278  Chapter  IX 

development  of  acquired  immunity,  but  in  this  case  the  properties 
of  the  fluids  of  the  body  are  but  little  in  evidence  or  they  may  be 
even  nil.  Let  us  return  to  the  example  of  the  Trypanosoma  of  the 
rat  which  excites  in  vaccinated  animals  a  protective  and  weakly 
agglutinative  power  of  the  serum.  This  fluid,  however,  is  usually 
found  to  be  incapable  even  of  rendering  the  flagellated  parasites 
motionless. 

The  question  of  immunity  against  malaria  has  been  much  dis- 
[292]  cussed.  It  is  well  known  that  a  first  attack  of  this  disease,  so  far 
from  conferring  any  immunity  of  the  slightest  durability,  leaves  a 
certain  predisposition  to  another  attack.  In  spite  of  this  the  study 
of  malaria  in  various  countries  and  in  individuals  belonging  to 
different  races  has  demonstrated  that  there  does  indeed  exist  a 
certain  degree  of  acquired  immunity  against  this  disease.  During 
recent  years  Koch1  has  paid  special  attention  to  this  subject  and  has 
furnished  us  with  very  valuable  data,  based  especially  on  a  com- 
parative study  of  the  blood  of  children  and  adults.  The  frequency 
of  Laveran's  parasite  in  the  former  and  its  rarity  in  the  latter,  have 
led  him  to  the  conclusion  that  infantile  malaria  sets  up  an  immunity 
which  persists  in  the  adult.  Moreover,  it  has  been  established  that 
in  malarial  countries  the  indigenous  inhabitants  exhibit  an  attenuated 
form  of  the  disease,  unaccompanied  by  acute  attacks,  but  with  phe- 
nomena that  are  chronic  and  very  slow  in  development. 

In  spite  of  the  existence  of  a  certain  degree  of  acquired  im- 
munity against  malaria,  all  attempts  to  demonstrate  any  protective 
action  of  the  serum  have  been  fruitless.  Celli2,  indeed,  injected,  as  a 
preventive,  the  blood  serum  of  individuals  who  had  recovered  from 
malaria  or  of  others  who  were  bled  during  the  period  of  defer- 
vescence following  an  acute  crisis  of  this  disease,  but  in  every 
instance  these  injections  were  found  to  be  useless  in  preventing  an 
attack  of  malaria. 

We  can  readily  understand  that  in  a  disease  which  is  exclusively 
human,  such  as  malaria,  it  has  not  been  possible  to  perform  a 
sufficient  number  of  experiments  to  decide  the  question  of  the 
protective  property  of  the  blood.  In  this  respect 'we  shall  have 

1  Deutsche  med.  Wchnschr.,  Leipzig,  1900,  S.  781. 

2  "  La  Malaria  secondo  le  nuove  recherche,"  Roma,  1899,  p.  86  [translated  into 
English  by  Byre  from  the  2nd  Italian  edition  under  the  title  "  Malaria  according  to 
the  new  researches,"  London,  1900].    "  Die  Malaria  "  [German  translation  of  same]  in. 
Behring's  "  Beitrage  z.  exper.  Therapie,"  1900,  Bd.  i,  Hft.  3. 


Acquired  immunity  against  micro-organisms      279 

greater  chance  of  obtaining  satisfactory  data  if  we  direct  our  atten- 
tion to  some  analogous  disease  attacking  one  of  the  lower  animals. 
Such  a  disease  we  have  in  Texas  fever,  occurring  in  the  Bovidae, 
as  the  result  of  the  action  of  an  animal  parasite,  Piroplasma 
bigeminwn,  which  invades  the  red  blood  corpuscles  much  as  Laveran's 
parasite  invades  those  of  the  human  subject. 

As  mentioned  in  the  preceding  chapter,  Smith  and  Kilborne 
and  Koch  have  demonstrated  that  the  Bovidae  may  acquire  a 
real  immunity  against  Texas  fever.  Xicolle  and  Adil  Bey1  at  [-293] 
Constantinople  found  indigenous  races  that  exhibited  a  remarkable 
immunity  against  the  Piroplasma.  Having  demonstrated  this  fact 
the  idea  occurred  to  them  to  inoculate  these  refractory  cattle  with 
very  large  quantities  of  virulent  blood  and  to  make  use  of  the  serum 
from  animals  so  treated  for  the  prevention  of  infection  in  susceptible 
races  of  Bovidae.  This  experiment  gave  negative  results.  Lignieres2 
elaborated  a  special  method  of  vaccinating  susceptible  Bovidae  and 
was  successful  in  obtaining  very  encouraging  results.  A  commission 
of  veterinary  surgeons  from  Alfort3  appointed  to  verify  these  observ- 
ations came  to  the  conclusion  that  "the  vaccination  as  carried  out 
by  Lignieres  was  absolutely  effective." 

Lignieres  also  carried  out  researches  on  the  protective  power  of 
the  blood  serum  of  his  immunised  cattle.  In  a  communication  to 
the  International  Congress  of  Medicine,  held  in  Paris  in  1900,  he 
stated  that  the  injection  of  several  hundred  cubic  centimetres  of  this 
fluid  did  not  protect  normal  animals  against  infection.  We  must 
conclude,  therefore,  that,  here  also,  we  have  another  example  of 
acquired  immunity  unaccompanied  by  the  presence  of  any  protective 
property  of  the  blood  fluid. 

These  results  have  received  confirmation  from  a  most  authoritative 
source.  Nocard  has  kindly  communicated  to  me  the  fact  that  he  has 
tried  in  vain  to  confer  immunity  on  normal  dogs  into  which  he  has 
injected  blood  serum  coming  from  dogs  that  had  recovered  from  the 
disease  produced  by  a  haematozoon  closely  allied  to  that  of  Texas 
fever  or  serum  from  sheep  immunised  with  blood  from  the  affected 
dogs. 

Looking  at  the  data  we  have  just  summarised  as  a  whole,  we  are 

1  Ann.  de  FInst.  Pasteur,  Paris,  1899,  t  xm,  p.  343. 

2  "La  'Tristeza'  ou  Malaria  bovine  dans  la  Republique  Argentine,"  Buenos 
Ayres,  1900,  p.  142. 

3  Bull.  Soc.  centr.  de  med,  veterin.,  Paris,  1900,  seances  des  12  et  26  juillet. 


280  Chapter  IX 

compelled  to  recognise  that,  on  the  one  hand,  the  protective  power  of 
the  body  fluids  may  coincide  with  a  susceptibility  to  the  corre- 
sponding micro-organism,  and  that,  on  the  other,  real  acquired 
immunity  may  exist  without  any  manifestation  of  this  humoral 
property,  especially  as,  even  in  immunised  animals,  the  acquired 
immunity  often  persists  longer  than  does  this  property.  It  must 
be  accepted  then,  that,  in  this  immunity,  there  exists  something 
other  than  the  powers  of  the  fluids  of  the  body,  that  is  to  say,  the 
factor  which  plays  the  predominant  part  is  to  be  sought  for  in  the 
[294]  cellular  elements.  We  need  only  recall  the  many  facts  collected  in 
the  preceding  chapter  to  be  convinced  that  in  acquired  immunity 
phagocytosis  is  the  most  constant  and  most  general  phenomenon. 
We  find  it  in  cases  where  the  humoral  properties  are  the  most 
marked,  as  well  as  in  those  in  which  they  are  only  slightly  developed 
or  are  entirely  absent.  We  need  not  again  discuss  Pfeiffer's  phe- 
nomenon analysed  in  the  preceding  chapter.  It  is  sufficient  to 
mention  that  this  example  of  the  extracellular  destruction  of  micro- 
organisms only  occurs  under  limited  and  special  conditions.  It  is 
observed  only  in  cases  where  the  injection  is  made  into  a  situation 
rich  in  leucocytes  which  undergo  phagolysis  as  a  result  of  the  sudden 
change  brought  about  in  their  conditions  of  existence.  Further,  this 
phenomenon  is  observed  only  in  connection  with  micro-organisms 
that  are  slightly  resistant  to  the  influence  of  the  microcytases.  In 
those  cases  in  which  we  meet  with  Pfeiffer's  phenomenon,  we  also 
meet  with  a  widely  extended  phagocytic  reaction. 

This  reaction  is  most  pronounced  where  the  properties  of  the 
body  fluids  are  only  slightly  developed  or  are  absent.  The  study 
of  acquired  immunity  against  anthrax  provides  us  with  a  very 
convincing  proof  of  this.  We  have  already  cited  the  example  of 
vaccinated  rabbits  and  rats  in  which  phagolysis  is  incomparably 
greater  than  in  the  susceptible  control  animals  which  contract  a  fatal 
anthrax.  This  rule  is  general.  It  is  confirmed  in  the  vaccinated 
sheep  and  guinea-pig.  The  absence,  or  feeble  development,  of  the 
protective  power  of  the  blood  or  of  the  other  humoral  properties  in 
no  way,  then,  prevents  the  considerable  change  which'  is  set  up  in  the 
phagocytes  of  animals  that  have  acquired  immunity  against  anthrax. 
The  negative  chemiotaxis  of  the  leucocytes,  so  marked  in  susceptible 
animals,  is  modified  into  positive  chemiotaxis  as  the  result  of 
vaccination.  This  fact,  one  of  fundamental  importance,  was  first 
demonstrated  for  the  immunity  against  anthrax,  later  being  ex- 


Acquired  immunity  against  micro-organisms      281 

tended  to  other  micro-organisms.  Massart1  studied  the  general 
subject  and  collected  a  series  of  data  which  led  him  to  say  that 
"  vaccination  effects  an  education  of  the  leucocytes  ;  these  latter 
become  so  adapted  that  they  can  approach  the  virulent  micro- 
organisms.'' The  best  method  of  forming  an  estimate  of  the  change 
which  the  leucocytes  undergo  is  by  injecting  subcutaneously  very  [295] 
virulent  micro-organisms  capable  of  setting  up  a  generalised  in- 
fection. The  anthrax  bacillus,  Gamaleia's  vibrio,  the  streptococci  and 
the  cocco- bacilli  of  swine  and  fowl  cholera  are  very  suitable  for  such 
study.  These  micro-organisms,  when  inoculated  subcutaneously  into 
susceptible  animals,  set  up  a  very  slight  local  reaction  or  none  at  all, 
in  the  form  of  an  exudation  of  transparent  fluid  almost  entirely 
without  leucocytes.  The  micro-organisms  grow  freely  in  these  ex- 
udations and  soon  invade  the  animal.  In  vaccinated  animals  the 
local  reaction  is  more  marked  and  the  exudation,  very  rich  in 
leucocytes,  is  poor  in  fluid ;  the  micro-organisms  remain  free  for  a 
very  short  time,  being  soon  ingested  by  the  leucocytes.  Their  de- 
struction, inside  these  cells,  takes  a  longer  or  shorter  time  according 
to  circumstances ;  but  in  the  end  it  is  always  complete. 

The  difference  as  regards  phagocytic  reaction  between  susceptible 
and  vaccinated  animals,  such  as  I  have  just  described,  has  been 
generally  recognised  by  many  observers.  A  few  opponents  are  still 
found,  however,  who  consider  that  they  are  justified  in  affirming  that 
the  negative  chernio taxis  of  the  susceptible  animal  does  not  exist  and 
that,  consequently,  vaccination  can  in  no  way  change  it  into  positive 
chemiotaxis.  Werigo  made  himself  the  spokesman  of  this  view, 
which  he  has  maintained  in  several  papers2.  Instead,  however,  of 
introducing  the  virulent  micro-organisms  into  the  subcutaneous 
tissue  of  susceptible  animals  he  injected  them  directly  into  the  veins. 
Using  cultures  of  the  anthrax  bacillus  and  of  the  cocco-bacillus  of 
fowl  cholera  he  injects  these  into  the  venous  system  of  normal 
rabbits.  The  animals  soon  die  from  general  infection.  If,  however, 
these  animals  are  killed  shortly  after  inoculation,  it  is  found  on 
examination  of  sections  that  many  of  the  micro-organisms  have 
been  ingested  by  the  leucocytes.  Werigo  concludes  from  these  facts 
that  in  the  higher  animals  the  chemiotaxis  is  always  positive  ;  but 
that  it  ends  in  the  destruction  of  the  micro-organisms  in  the  vaccin- 

1  Ann.  de  Vlnst.  Pasteur,  Paris,  1892,  t.  vi,  p.  321. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1894,  t.  vm,  p.  1 ;  Arch,  de  med.  exper.,  Paris, 
1898,  t.  x,  p.  725;  Arch,  russes  de  Path.  &c.,  St  Petersb.,  1898. 


282  Chapter  IX 

ated  animals,  never  bringing  about  this  result  in  susceptible  animals. 
Taking  all  the  data  on  this  question  into  consideration,  it  is  easy  to 
convince  oneself  that  this  view  cannot  be  accepted  as  correct,  for  not 
[296]  only  the  definite  phenomena  observed  below  the  skin  but  also  the  no 
less  demonstrative  process  appearing  in  the  peritoneal  cavity  prove 
most  clearly  the  existence  of  this  negative  chemiotaxis  of  the 
leucocytes.  I  need  only  recall  Bordet's  experiment  on  the  fate  of 
streptococci  and  Proteus  vulgaris  when  injected  together  into  the 
peritoneal  cavity  of  guinea-pigs.  Whilst  the  Proteus  bacilli  at  the 
end  of  a  very  short  time  are  all  ingested  by  the  leucocytes,  the 
streptococci  remain  free  in  the  peritoneal  fluid  up  to  the  death  of 
the  animal.  The  leucocytes  which  exhibit  a  positive  chemiotaxis  as 
regards  the  former,  manifest  a  negative  chemiotaxis  as  regards  the 
streptococci. 

In  spite  of  the  great  force  of  these  arguments,  the  discovery  of  a 
means  of  reconciling  the  results  obtained  from  the  inoculation  of 
micro-organisms  subcutaneously  or  into  the  peritoneal  cavity,  with 
those  observed  after  they  had  been  injected  into  the  blood  vessels 
would  be  of  great  interest,  and  Zilberberg  and  Zeliony x  have  under- 
taken a  series  of  experiments  with  this  object.  Following  Werigo 
they  made  use  of  the  cocco-bacilli  of  fowl  cholera,  and  found,  in 
accordance  with  his  observations,  that  the  intravenous  injection  of 
these  organisms,  obtained  from  cultures  in  nutrient  media,  causes  a 
very  marked  phagocytosis  of  the  cocco-bacilli.  When,  however,  they 
injected  into  the  veins  of  rabbits  cocco-bacilli  that  had  been  grown  in 
the  peritoneal  fluid  of  other  rabbits,  they  found  the  micro-organisms 
free  in  the  blood  plasma  and  observed  only  a  very  restricted  phagocy- 
tosis in  the  microphages  of  the  liver.  It  follows  from  these  experiments 
that  the  ingestion  of  the  cocco-bacilli,  in  Werigo's  experiments,  was 
dependent  on  the  presence  of  a  large  number  of  attenuated  micro- 
organisms which  were  present  in  the  cultures  that  he  employed  for 
his  injections.  Alongside  these  organisms,  slightly  or  not  virulent, 
were  others,  endowed  with  their  normal  pathogenic  activity  and 
quite  numerous  enough  to  set  up  a  fatal  infection.  When  Zilberberg 
and  Zeliony  replaced  cultures  on  agar  by  the  peritoneal  exudation 
which  contained  virulent  cocco-bacilli  almost  exclusively,  the  phago- 
cytosis in  rabbits,  injected  into  the  veins,  was  found  to  be  almost 
suppressed.  With  the  object  of  establishing  whether  the  absence  of 
the  phagocytic  reaction,  in  this  case,  really  depended  on  negative 
1  Ann.  de  I'Inst.  Pasteur,  Paris,  1901,  t.  xv,  p.  615. 


Acquired  immunity  against  micro-organisms      283 

chemiotaxis  on  the  part  of  the  leucocytes,  the  above  cited  observers 
performed  the  following  experiment.  They  injected  into  the  vein  of  [297] 
a  rabbit,  already  affected  with  a  generalised  infection  by  the  cocco- 
bacillus  of  fowl  cholera,  an  innocuous  culture  of  a  saprophytic 
staphylococcus.  Post-mortem  examination  showed  that  these  cocci 
were  almost  entirely  ingested  by  the  same  phagocytes  which  refused 
so  energetically  to  seize  the  cocco-bacilli.  This  experiment,  analogous 
to  that  of  Bordet  on  streptococcus  and  Proteus,  compels  us  to  reject 
Werigo's  conclusions  as  to  the  absence  of  negative  chemiotaxis  in  the 
phagocytes  of  the  higher  animals.  I  ought  to  add  that  the  work  of 
Zilberberg  and  Zeliony  was  in  part  executed  in  my  laboratory  so  that 
I  was  able  to  convince  myself  by  ocular  demonstration  of  the  com- 
plete accuracy  of  their  statements. 

Independently  of  these  observers  and  even  before  their  work  ap- 
peared, Th.  Tchistovitch1  published  an  interesting  study  on  the  same 
question.  He  injected  very  virulent  streptococci  into  the  ear  vein  of 
rabbits.  These  micro-organisms  set  up  a  generalised  and  fatal  infec- 
tion in  which  phagocytosis  was  completely  absent  or  nearly  so.  Here 
again  was  manifested  a  negative  chemiotaxis  of  the  phagocytes,  which, 
henceforth,  could  no  longer  be  questioned. 

In  certain  infective  diseases  terminating  fatally  a  very  marked 
phagocytosis  is  observed  even  in  susceptible  animals.  The  most 
typical  example  of  this  is  furnished  by  swine  erysipelas  and  mouse 
septicaemia.  We  know  from  the  researches  of  Koch2,  followed  by 
those  of  Loeffler3,  Schtitz4  and  others,  that  in  animals  which  have 
died  from  these  two  diseases  the  leucocytes  are  gorged  with  small 
specific  bacilli.  A  method  of  vaccinating  animals  against  the  micro- 
organism of  swine  erysipelas  was  worked  out  by  Pasteur  and 
Thuillier5  and  was  afterwards  studied  by  many  observers.  Thanks  to 
this  method  it  has  been  possible  to  demonstrate  the  phenomena  which 
may  be  observed  in  vaccinated  animals  (especially  rabbits).  Here 
also  a  phagocytosis  takes  place,  even  more  rapid  and  more  complete 
than  in  susceptible  animals.  What  is  more  important,  the  intra- 
cellular  digestion  of  the  ingested  bacilli  is  followed  by  the  total  L2981 

1  Ann.  de  VInst.  Pasteur,  Paris,  1900,  t.  xiv,  p.  802. 

"Untersuehungen  iiber  die  Aetiologie  der  Wundinfectionskrankheiten,"  Leipzig, 
1878.  [Translated  into  English  in  the  New  Sydeuham  Society's  Series,  London, 
1880,  Vol.  LXXXVIII,  under  the  title  "On  Traumatic  Infective  Diseases."] 

3  Arb.  a.  d.  K.  Gsndhtsamt.,  Berlin,  1885,  Bd.  I,  S.  46. 

4  Arb.  a.  d.  K.  Gsndhtsamt.,  Berlin,  1885,  Bd.  i,  S.  57. 

5  Compt.  rend.  Acad.  d.  sc.,  Paris,  1883,  t  xcvn,  p.  1163. 


284  Chapter  IX 

destruction  of  the  micro-organisms  in  the  vaccinated  animals,  though 
in  the  normal  animals  this  digestion  is  very  imperfect. 

The  acquisition  of  immunity  against  micro-organisms  is,  therefore, 
due  not  only  to  the  change  from  negative  to  positive  chemiotaxis,  but 
also  to  the  perfecting  of  the  phagocytic  and  digestive  powers  of  the 
leucocytes — a  general  superactivity  and  adaptation  of  the  phagocytic 
reaction  of  the  immunised  animal  is  produced.  This  conclusion, 
based  upon  a  large  number  of  well-established  facts  and  in  complete 
harmony  with  the  whole  of  the  data  at  our  disposal  concerning 
acquired  immunity,  has  been  attacked  by  Denys  and  Leclef l  in  their 
work  on  the  streptococcus.  They  base  their  opposition  upon  experi- 
ments made  in  vitro  on  the  action  of  serums  and  leucocytes  on  this 
micro-organism.  They  have  compared  the  bactericidal  power  of 
mixtures  of  the  serums  of  normal  and  of  vaccinated  rabbits  with 
leucocytes  isolated  from  exudations  from  these  two  groups  of 
animals.  The  leucocytes,  whether  derived  from  normal  or  from 
vaccinated  rabbits,  when  mixed  with  normal  serum  were  equally 
incapable  of  ingesting  and  destroying  the  streptococci.  When  mixed 
with  blood  serum  from  vaccinated  rabbits,  however,  the  two  kinds  of 
leucocytes  exhibited  a  very  marked  phagocytic  reaction.  Denys  and 
Leclef  conclude  from  this  that  phagocytosis,  although  an  important 
factor  in  immunity,  plays  merely  a  secondary  part  and  is  dependent 
on  the  humoral  properties.  The  experiments  and  views  of  these 
two  observers  have  been  generally  received  by  the  partisans  of 
the  bactericidal  theory  of  the  body  fluids  as  an  actual  proof  of 
this  theory.  We  cannot  agree.  Researches  extending  over  a  long 
period  have  shown  us  that  the  study  of  phagocytosis  in  vitro  can 
give  only  a  very  inexact  and  imperfect  idea  of  the  course  of  the 
phenomena  in  the  living  animal.  Usually  the  leucocytes  taken  from 
the  exudations,  although  amoeboid,  no  longer  fulfil  their  phagocytic 
functions  at  a  time  when  in  the  animal  they  would  ingest  micro- 
organisms with  the  greatest  rapidity.  As  a  general  rule,  existence 
outside  the  living  body  weakens  them  very  considerably.  But  in  some 
cases,  rare  it  is  true,  the  leucocytes  although  inactive  in  the  animal 
[299]  exhibit  intense  phagocytosis  when  introduced  into  a -hanging  drop  of 
fluid  from  an  exudation  or  even  of  urine.  In  any  case  it  is  very 
hazardous  to  infer  from  phenomena  which  appear  under  these 
artificial  conditions  what  takes  place  in  the  living  animal.  The  value 
of  the  experiments  of  Denys  and  Leclef  is  still  further  marred  by 
1  La  Cellule,  Lierre  et  Lou  vain,  1895,  t  xi,  p.  177. 


Acquired  immunity  against  micro-organisms      285 

the  fact  that  they  mixed  the  leucocytes  with  blood  serum.  They 
appear  to  have  lost  sight  of  the  fact  that  this  fluid  is  far  from 
corresponding  to  that  which  bathes  the  leucocytes  in  the  living 
animal.  The  serums  contain  leucotoxin  in  greater  or  less  quantity 
and  it  is  not  to  be  wondered  at  that  the  leucocytes  when  mixed  with 
normal  rabbit's  serum  should  perish  very  rapidly.  Further,  the 
serum  of  vaccinated  rabbits  is  agglutinative  (this  fact,  however,  was 
not  sufficiently  elucidated  in  1894  when  the  researches  of  Denys  and 
Leclef  were  made)  and  the  clumping  of  streptococci  might  simulate 
their  destruction.  In  a  word,  the  experiments  of  these  observers 
have  been  carried  out  under  such  conditions  that  it  is  impossible  to 
base  upon  them  a  refutation  of  data  obtained  in  the  living  animal. 
Moreover,  in  the  description  of  the  phenomena  which  appear  in  the 
subcutaneous  tissue  of  rabbits  inoculated  with  the  streptococcus, 
Denys  and  Leclef  provide  us  with  arguments  against  their  own 
view. 

These  observers  introduce  the  same  quantity  of  streptococci 
below  the  skin  of  the  ear  of  normal  and  of  vaccinated  rabbits.  In 
the  first  there  is  soon  produced  a  very  marked  oedema  of  the  ear,  in 
which  may  be  seen  a  number  of  streptococci  and  of  leucocytes  that 
have  not  ingested  any  micro-organisms.  In  the  second  the  oedema 
does  not  develop,  but  at  the  seat  of  invasion  a  number  of  leucocytes 
come  up  and  these  soon  ingest  the  streptococci.  As  we  see,  the 
phenomena  manifest  themselves  here  just  as  they  do  with  the  anthrax 
bacillus  and  many  other  micro-organisms  when  under  analogous 
conditions.  Denys  and  Leclef,  indeed,  recognise  that,  below  the  skin 
of  the  ear  of  vaccinated  rabbits,  the  small  quantity  of  exudation 
fluid  is  not  sufficient  to  enable  us  to  accept  it  as  capable  of  exerting 
any  considerable  influence  as  regards  humoral  properties.  Neverthe- 
less, they  think  that  the  "  serum  "  of  this  fluid  may  exercise  a  certain 
action,  but  they  furnish  no  proof  of  this,  and  seem  to  ignore  the  fact 
that  the  plasma  of  the  subcutaneous  exudation  is  far  from  being 
identical  with  blood  serum  obtained  outside  the  animal.  At  present 
it  is  well  known  that  this  latter  fluid  contains  cytases  which  are[300j 
absent  from  the  plasmas.  Now,  the  feeble  bactericidal  action,  if  this 
really  exists  as  regards  the  streptococcus,  must  be  attributed  to  the 
microcytase  which  has  escaped  from  the  leucocytes  at  the  time  of  the 
preparation  of  the  serum. 

To  sum  up,  the  example  studied  by  Denys  and  Leclef  clearly 
comes   under  the  general  law  of  phagocytic   reaction   in  acquired 


286  Chapter  IX 

immunity  against  micro-organisms.  It  is  impossible  to  deny  that 
the  superactivity  of  the  phagocytes  which  is  always  found  in  this 
immunity,  although  readily  observed,  cannot  be  demonstrated  in  a 
rigorous  fashion  outside  the  fluids  which  bathe  the  cells.  There  are, 
however,  very  important  analogies  which  may  be  invoked  in  favour 
of  this  thesis.  We  have  already  cited  in  our  fifth  chapter  Delezenne's 
experiments  on  the  digestion  of  gelatine  by  the  leucocytes  of  the 
dog,  which  show  in  the  most  demonstrative  fashion  that  these  cells 
accustom  themselves  to  bring  about  this  digestion  more  and  more 
quickly  and  this  quite  independently  of  any  humoral  influence. 

For  some  time  past  there  has  been  no  doubt  as  to  the  funda- 
mental fact  that  the  phagocytes  in  immunised  animals  seize  and 
destroy  living  micro-organisms.  Several  attempts  have  been  made 
to  show  that  such  destruction  of  these  bacteria  takes  place  solely 
by  the  body  fluids,  and  that  the  phagocytes  intervene  only  as 
"  scavengers "  to  carry  off  the  dead  bodies  of  the  micro-organisms. 
The  numerous  observations,  described  in  the  preceding  chapter, 
absolve  us  from  again  entering  into  a  discussion  of  this  question. 
Moreover,  the  majority  of  these  opponents  now  recognise  that  micro- 
organisms are  ingested  in  a  living  state  by  the  phagocytes  of  immu- 
nised animals.  Some,  however,  have  expressed  the  opinion  that 
these  living  micro-organisms,  before  becoming  the  prey  of  the  phago- 
cytes, must  undergo  some  preliminary  attenuation  of  virulence 
through  the  action  of  the  body  fluids.  Hence  the  theory  of  the 
attenuating  power  of  the  fluids  of  the  body,  maintained  especially  by 
Bouchard  and  his  pupils.  During  the  course  of  our  exposition  of  the 
facts  concerning  acquired  immunity,  we  have  several  times  had 
occasion  to  speak  of  the  virulence  of  micro-organisms  in  the  im- 
munised animal.  Here,  therefore,  we  may  confine  ourselves  to  a  brief 
summary  of  the  observations  collected  on  this  point. 

Having  observed  that  the  anthrax  bacillus,  when  developed  in  the 
blood  of  immunised  sheep,  was  incapable  of  giving  fatal  anthrax  to 
[301]  rabbits,  I  expressed1  the  opinion  that  under  these  conditions  its 
virulence  had  become  attenuated.  Later,  analogous  changes  were 
shown  by  Charrin2  in  the  Bacillus  pyocyaneus  when  cultivated  in  the 
serum  of  immunised  animals.  Bouchard3,  generalising  on  these  data, 
arrived  at  the  following  theory  of  vaccination.  "  The  inoculation  of 

1  Ann.  de  VInst.  Pasteur,  Paris,  1887, 1. 1,  p.  42. 

2  Compt.  rend.  Soc.  de  biol.,  Paris,  1889-1891. 

3  "  Essai  d'une  theorie  de  1'infection."    Berlin,  1890. 


Acquired  immunity  against  micro-organisms      287 

a  strong  virus  into  a  vaccinated  animal  is  equivalent  to  the  inocula- 
tion of  an  attenuated  virus.  The  attenuation,  however,  instead  of 
being  done  beforehand  in  the  laboratory,  is  brought  about  in  the 
tissues  of  the  vaccinated  animal "  (p.  18).  Charrin  and  Roger1  up- 
held this  view,  and  the  latter  offered  several  new  arguments  in 
support  of  it.  He  observed  that  animals  inoculated  with  pneumo- 
cocci  and  streptococci  grown  in  the  blood  serum  of  vaccinated 
animals,  contracted  a  transient  and  benign  disease  merely,  whilst 
the  control  animals,  inoculated  with  the  same  micro-organisms, 
cultivated  in  normal  serum,  always  died  from  generalised  in- 
fection. 

The  discovery  of  the  protective  property  of  serums  has  thrown  a 
new  light  upon  these  experiments.  We  must  now  ask  ourselves: 
Does  the  innocuousness  of  micro-organisms  depend  not  on  the  at- 
tenuation of  the  virus,  but  rather  on  the  protective  action  of  the 
serum  itself?  When,  in  the  course  of  my  researches  on  the  Gentilly 
cocco-bacillus,  I  found  that  this  organism,  cultivated  in  the  serum 
of  vaccinated  rabbits,  became  much  less  pathogenic  than  when  it 
was  grown  in  the  serum  of  normal  rabbits,  I  set  myself  to  answer 
this  question.  Simple  filtration  through  paper  was  sufficient  to  rid 
the  organism  of  the  serum  in  which  it  had  grown.  The  inoculation 
of  cocco-bacilli  thus  treated  proved  at  once  that  their  virulence  was 
in  no  degree  modified,  and  that  it  was  the  intervention  of  the  serum 
that  prevented  the  micro-organism  from  setting  up  the  rapidly  fatal 
disease.  Issaeff 2,  who,  in  my  laboratory,  carried  out  the  investigation, 
was  able  to  extend  this  to  the  pneumococcus.  He  obtained  agglu- 
tinated cultures  in  the  serum  of  vaccinated  rabbits,  and  he  compared 
their  activity  by  injecting  them  (1)  with,  and  (2)  without  their  culture  [302] 
medium.  The  difference  was  very  marked.  In  the  first  case  the 
infection  produced  was  much  slower  in  its  course  than  in  the  second. 
The  virulence  of  the  washed  pneumococci  was  found  to  be  the  same 
whether  they  came  from  a  culture  in  normal  serum  or  from  one  in 
immunised  serum.  Sanarelli3  obtained  the  same  result  with  Gama- 
leia's  vibrio.  The  vibrios  when  grown  in  the  serum  of  vaccinated 
guinea-pigs  proved  to  be  very  virulent  so  soon  as  they  were  freed 
from  the  fluid  in  which  they  were  grown.  Later,  similar  demon- 

1  Charrin,  Oompt.  rend.  Soc.  de  biol.,  Paris,  1890,  pp.  203,  332 ;  Roger,  ibid., 
1890,  p.  573,  and  Rev.  gen.  d.  sc.  pures  et  appliq.,  Paris,  1891,  p.  410. 

2  Aim.  de  VInst.  Pasteur,  Paris,  1893,  t.  vn,  p.  273. 

3  Ann.  de  VInst.  Pasteur,  Paris,  1893,  t.  vn,  p.  231. 


288  Chapter  IX 

st rations  were  given  by  Bordet1  and  Mesnil2  with  respect  to  strepto- 
cocci and  to  the  bacilli  of  swine  erysipelas.  We  must,  then,  conclude 
that  we  have  here  to  do  with  a  general  law.  Some  experiments 
made  by  de  Nittis3  might  seem  to  indicate  an  exception  to  such  a 
law.  He  observed  that  anthrax  bacilli  when  grown  in  the  serum  of 
vaccinated  pigeons  lost  a  part  of  their  virulence.  It  must  not  be 
forgotten,  however,  that  he  grew  his  cultures  under  special  con- 
ditions ;  the  bacillus  was  grown  for  several  days  at  42°  C.,  this  in 
itself  being  quite  sufficient  to  bring  about  a  certain  attenuation  of 
virulence. 

The  theory  of  the  attenuating  action  of  the  body  fluids,  based  on 
the  attenuation  of  the  viras  in  the  serum  of  vaccinated  animals,  can 
no  longer  be  maintained,  as  it  is  a  well-established  fact  that  the 
serum,  obtained  outside  the  body,  is  a  fluid  differing  in  character  and 
properties  from  the  plasma  of  the  living  animal.  We  have  seen  up 
to  what  point  this  demonstration  has  shaken  the  theory  of  the 
bactericidal  action  of  the  body  fluids. 

It  cannot  be  doubted  that  a  micro-organism  may  undergo  a  certain 
weakening  in  virulence,  as  well  as  in  certain  other  functions,  in  the 
body  of  the  animal  that  has  acquired  immunity.  But  the  question 
must  be  put :  Is  this  effect  obtained  as  the  result  of  humoral  or  of 
cellular  action?  As  a  general  rule,  exudations  obtained  from 
vaccinated  animals,  and  containing  living  micro-organisms,  are  found 
to  be  virulent  when  inoculated  directly  into  susceptible  animals. 
This  fact  was  established  by  Pasteur4  when  he  first  carried  out  his 
researches  on  acquired  immunity  against  fowl  cholera.  He  showed 
that  the  exudations  of  vaccinated  fowls  set  up  a  fatal  disease  in 
[303]  normal  fowls,  without  there  being  the  least  evidence  of  any  attenu- 
ation of  the  micro-organism.  The  same  applies  to  the  Gentilly 
cocco-bacillus  and  to  the  anthrax  bacillus  in  a  very  great  majority 
of  examples.  De  Nittis  observed  that  the  exudations  of  immunised 
pigeons  produced  a  fatal  infection  in  the  guinea-pig  and  in  the  mouse. 
In  the  immunised  guinea-pig,  on  the  other  hand,  he  found  that  the 
exudations  soon  became  innocuous  for  these  animals.  This  alteration, 
however,  must  be  attributed  not  to  the  body  fluids  (which  exhibit  no 
protective  or  attenuating  power)  but  to  the  action  of  the  cells. 

1  Ann.  de  I'Inst.  Pasteur,  Paris,  1897,  t.  xi,  p.  177. 

2  Ann.  de  I'Inst.  Pasteur,  Paris,  1898,  t  xn,  p.  481. 

3  Ann.  de  I'Inst.  Pasteur,  Paris,  1901,  t.  xv,  p.  769. 

4  Compt.  rend.  Acad.  d.  sc.,  Paris,  1880,  t.  xo,  p.  1033. 


Acquired  immunity  against  micro-organisms      289 

"With  the  object  of  gaining  some  idea  of  the  changes  that  the 
micro-organisms  undergo  in  the  immunised  animal,  Vallee1  carried 
out  a  series  of  experiments  on  rabbits  vaccinated  against  the  bacillus 
of  swine  erysipelas.  He  enclosed  these  bacilli  in  sacs  of  collodion 
which  he  introduced  into  the  peritoneal  cavity  of  susceptible  rabbits 
and  of  others  that  were  hyperimmunised.  The  bacillus  developed  well 
in  both  cases.  It  gave  homogeneous  non-agglutinated  cultures  in  the 
sacs  placed  in  normal  animals,  whilst  in  the  sacs  introduced  into 
the  peritoneal  cavity  of  hyperimmunised  rabbits  the  bacilli  grew  into 
agglutinated  filaments.  This  proves  that  the  wall  of  the  sacs  per- 
mitted of  the  passage  of  the  active  substances  elaborated  in  the 
immunised  animal.  Different  from  the  point  of  view  of  agglutination, 
the  cultures  likewise  exhibited  a  considerable  difference  in  their 
pathogenic  activity.  The  cultures  developed  in  the  sacs  in  hyper- 
immunised rabbits  were  found  to  be  much  more  virulent  than  those 
grown  in  the  sacs  in  control  animals.  This  augmentation  of  virulence 
depends,  probably,  on  the  influence  of  the  active  substances  which 
pass  through  the  Avails  of  the  sacs.  In  any  case,  this  experiment 
affords  further  confirmation  of  the  impossibility  of  maintaining  the 
theory  of  the  attenuation  of  micro-organisms  by  the  fluids  of  an 
animal  enjoying  acquired  immunity. 

Since  the  discovery  of  the  antitoxic  property  of  the  fluids  of  the 
body,  it  has  been  accepted  that  its  manifestation  was  indispensable 
for  the  acquisition  of  immunity.  It  was  thought  that  in  order  to  get 
rid  of  pathogenic  micro-organisms  the  animal  had  first  to  develop 
the  means  of  neutralising  their  toxins.  These  substances  once  pre- 
vented from  exerting  their  toxic  action,  the  micro-organisms  were 
left  without  their  weapon  of  attack  and  found  themselves  reduced 
to  the  condition  of  simple  saprophytes.  It  was  accepted,  therefore, 
that  an  effective  antitoxic  power  was  always  to  be  found  in  the  fluids 
of  animals  that  had  acquired  immunity.  Against  this  explanation,  [304] 
however,  are  certain  established  facts.  Chauveau2  had  observed  that 
Algerian  sheep,  whose  natural  immunity  was  further  strengthened 
by  considerable  doses  of  anthrax  bacilli,  exhibited  a  susceptibility 
to  injections  of  anthrax  blood  quite  as  marked  as  that  of  normal 
sheep.  The  immunity  against  the  virus,  then,  did  not  progress 
pari  passu  with  that  against  the  poison.  Later,  Charrin  and 

1  Compt.  rend.  Soc.  de  UoL,  Paris,  1899,  p.  432. 

2  Compt.  rend.  Acad.  d.  sc.,  Paris,  1880,  t.  xc,  p.  1526. 

B.  19 


290  Chapter  IX 

Gamaleia1  furnished  important  data  on  this  subject.  They  showed 
that  animals  vaccinated  against  the  Bacillus  pyocyaneus  and  the 
vibrios  of  Koch  and  Gamaleia  were  even  more  susceptible  to  intoxi- 
cation by  the  soluble  products  of  these  micro-organisms  than  were 
normal  animals  which  had  acquired  no  immunity  against  the  corre- 
sponding bacteria.  Shortly  afterwards  this  observation  was  confirmed 
by  Selander2,  in  his  work  on  hog  cholera,  carried  out  under  Roux's 
direction.  Babbits  vaccinated  against  the  cocco-bacillus  of  this  disease 
resisted  infection  by  the  virus,  but  died  as  a  result  of  the  exhibition 
of  the  same  doses  of  toxin  that  killed  normal  rabbits.  I3  was  able 
not  only  to  verify  this,  but  to  add  to  it  the  further  fact  that  the 
blood  serum  of  vaccinated  rabbits,  although  markedly  protective 
against  infection,  exercised  not  the  slightest  antitoxic  action. 

When,  later,  R.  Pfeifler  set  himself  to  study  the  immunity  of 
animals  against  the  cholera  vibrio,  he,  along  with  his  collaborators, 
was  able  to  furnish  numerous  data  confirming  the  hypothesis  that 
animals  thoroughly  vaccinated  against  this  vibrio  had  not  thereby 
become  more  resistant  to  its  toxin  and  that  their  anti-infective  serum 
exhibited  no  antitoxic  power.  These  results  have  been  confirmed 
repeatedly  and  must  be  regarded  as  fully  established. 

Von  Behring  here  recognised  a  general  law  which,  with  the  aid  of 
his  collaborators,  he  attempted  to  develop.  We  owe  to  him  the 
knowledge  that  the  susceptibility,  augmented  as  regards  the  toxins, 
of  animals  vaccinated  against  micro-organisms,  might  even  serve  in 
doubtful  cases  to  reveal  the  presence  of  their  bacterial  poisons. 
Culture  products  when  deprived  of  micro-organisms  often  set  up  no 
[305]  poisoning  in  normal  animals  susceptible  to  infection.  From  this  fact 
it  is  generally  concluded  that  the  toxin  is  not  present  in  the  products 
in  question.  But  animals  of  the  same  species  when  immunised 
against  infection  by  the  micro-organism,  owing  to  their  "hyper- 
susceptibility,"  react  much  more  delicately  and  allow  of  the  de- 
monstration of  the  presence  of  bacterial  poisons  in  fluids  inactive 
for  unvaccinated  animals. 

In  collaboration  with  Kitashima4,  von  Behring  immunised  guinea- 
pigs  against  the  diphtheria  bacillus,  and  demonstrated  that  two  or 
three  injections  of  diphtheria  toxin  were  quite  sufficient  to  render 

1  Compt.  rend.  Soc.  de  biol,  Paris,  1890,  p.  294. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1890,  t.  iv,  p.  563. 

3  Ann.  de  VInst.  Pasteur,  Paris,  1892,  t.  vi,  p.  295. 

4  Berl  klin.  Wchnschr.,  1891,  p.  157. 


Acquired  immunity  against  micro-organisms      291 

these  animals  refractory  to  infection  by  the  diphtheria  bacillus  though 
they  became  more  susceptible  to  intoxication.  Von  Behring  considers 
that  this  augmentation  of  susceptibility  to  the  diphtheria  poison  may 
be  a  means  of  rendering  the  local  reaction  of  the  living  elements  at 
the  point  of  introduction  of  the  bacilli  more  active. 

In  any  case,  it  is  beyond  question  that  acquired  immunity  against 
microbial  infection  is  quite  independent  of  the  resistance  against  the 
toxins  of  the  corresponding  micro-organism.  An  antitoxic  manifesta- 
tion of  any  kind,  therefore,  cannot  be  regarded  as  necessary  for  the 
development  of  immunity  against  the  micro-organism. 

Of  all  the  humoral  properties  developed  in  acquired  immunity 
against  micro-organisms,  the  fixative  power  and  the  protective  power 
are  the  most  constant.  It  might  naturally  be  suggested,  as  a  result 
of  this  observation,  that  these  two  powers  are  indispensable  for  the 
manifestation  of  phagocytosis  for  the  purpose  of  destroying  and  of 
ridding  the  animal  of  the  pathogenic  organisms.  It  is  quite  possible 
to  understand  how,  under  these  conditions,  the  idea  has  been  put 
forward  that  anti-infective  acquired  immunity  is  the  result  of  two 
different  factors :  in  the  first  place,  a  humoral  property  independent 
of  the  phagocytes  and,  in  the  second  place,  the  phagocytes  themselves. 
But  the  part  played  by  these  cells  cannot  be  accepted  as  purely 
secondary — a  view  which  has  been  advanced  and  defended  again  and 
again.  This  question  is  of  such  importance  that  it  is  reasonable  to 
ask  whence  come  the  humoral  properties,  such  as  the  fixative  power 
and  the  protective  power,  factors  of  such  far-reaching  influence  in 
anti-infective  immunity  ? 

Thanks  to  the  work  of  several  investigators  this  question  may  now  [306] 
be  answered.  Pfeiffer  and  Marx1  first  supplied  important  informa- 
tion concerning  the  origin  of  the  protective  property.  Into  rabbits 
they  made  subcutaneous  inoculations  of  cholera  vibrios,  killed  by 
heat  (70°  C.),  and  then  examined,  most  minutely,  the  protective  power 
of  the  blood  and  of  extracts  from  various  organs.  Examining,  sepa- 
rately, the  protective  power  of  the  serum  and  that  of  the  layer  of 
leucocytes  deposited  in  tubes,  Pfeiffer  and  Marx  were  unable  to  find 
any  marked  difference.  Nor  did  they  ever  obtain  any  definite  effect 
with  leucocytes  collected  from  pleuritic  exudations.  From  these 
observations  they  concluded  that  the  leucocytes  of  the  blood  could 
not  be  regarded  as  the  source  of  the  protective  substance  (or 

1  Ztschr  f.  Hyg.,  Leipzig,  1898,  Bd.  xxvn,  S.  272. 

19—2 


292  Chapter  IX 

"  cholera  antibody  ")•  At  a  period  when  the  serum  as  yet  exhibited 
an  insignificant  protective  power  or  none  at  all,  the  extract  from  the 
spleen  often  exerted  an  action  of  the  most  marked  character.  In  an 
experiment  in  which  the  rabbit  was  killed  48  hours  after  the  injection 
of  the  vibrios,  0*3  c.c.  of  the  serum  was  incapable  of  preventing  fatal 
infection  of  a  guinea-pig,  whereas  0'03  c.c.  of  an  extract  of  the  spleen 
exerted  a  marked  protective  effect.  From  this  and  similar  experi- 
ments, Pfeiffer  and  Marx  conclude  that  the  spleen  is  the  principal 
source  of  the  protective  substance.  In  order  to  verify  this  observation 
they  injected  killed  cholera  cultures  into  rabbits  which  had  previously 
been  deprived  of  their  spleens,  but  the  asplenic  rabbits  still  produced 
the  same  amount  of  protective  substance,  and  these  two  observers 
were  led  to  conclude  that  the  lymphatic  glands  and  the  bone-marrow 
might  also  serve  as  the  sites  of  origin  of  this  substance. 

It  is  only  during  the  first  few  days,  however,  that  these  organs 
exhibit  a  protective  power  greater  than  that  of  the  blood.  Three  or 
four  days  after  the  injection  of  the  vibrios  the  blood  serum  becomes 
richer  in  protective  substance;  the  organs  contain  much  less  of  it. 
This  condition  is  maintained  for  some  time,  after  which  the  blood  in 
turn  begins  to  get  impoverished. 

Pfeiffer  and  Marx  put  to  themselves  the  question :  Is  the  marked 
protective  power  of  the  spleen  due  to  the  production  of  preventive 
substance  by  this  organ,  or  is  it  to  be  explained  by  an  accumulation 
in  the  spleen  of  this  substance  manufactured  elsewhere?  With  the 
[307]  object  of  obtaining  an  answer  to  this  question  they  injected  protective 
serum  from  other  individuals  into  rabbits,  when  they  found  that  the 
protective  substance  showed  not  the  slightest  tendency  to  accumulate 
in  the  spleen.  These  authors  were  compelled  to  conclude,  therefore, 
that  the  spleen  and  other  haematopoietic  organs  (lymphatic  glands 
and  bone-marrow)  are  the  real  seats  of  the  production  of  the 
protective  substance.  We  may  add  that  these  organs  are  also  the 
phagocytic  organs  par  excellence,  that  is  to  say,  the  centres  which 
serve  not  only  for  the  development  of  phagocytes  but  which  contain 
a  large  number  of  the  adult  elements  capable  of  exercising  the 
phagocytic  function. 

Almost  simultaneously  with  Pfeiffer  and  Marx,  A.  Wassermann1,  in 
collaboration  with  Takaki,  undertook  similar  researches  on  the  origin 
of  the  substance  protective  against  the  typhoid  cocco-bacillus.  The 

!'  BerL  klin.  Wchnschr.,  1898,  S.  209. 


Acquired  immunity  against  micro-organisms      293 

outcome  of  this  work  was  that  "  it  was  the  bone-marrow,  the  spleen, 
and  the  lymphatic  system,  including  the  thymus  gland,  which  ex- 
hibited immunising  power  against  the  bacillus  of  typhoid  fever,  whilst 
the  other  organs,  the  blood,  brain,  spinal  cord,  muscles,  liver,  kidney, 
etc.,  did  not  at  this  stage  show  any  marked  specific  property." 

As  these  observations  on  the  production  of  protective  substance  in 
the  phagocytic  organs  was  one  of  essential  importance  in  connection 
with  the  problem  of  acquired  immunity,  I  asked  M.  Deutsch1,  working 
in  my  laboratory,  to  carry  out  a  series  of  experiments  on  this  subject. 
Using  guinea -pigs,  he  injected  into  the  peritoneal  cavity  cultures  of 
the  typhoid  bacillus  killed  by  heat  (66°  C.).  A  few  days  later  the 
serum  had  become  distinctly  protective.  At  this  stage,  and  even 
before  the  appearance  of  this  property  in  the  blood,  Deutsch  killed 
some  of  his  animals  and  carefully  measured  the  protective  power  of 
the  extract  of  the  various  organs.  He  began  by  confirming  the  result 
obtained  by  Pfeiffer  and  Marx  as  to  the  non-production  of  the  pro- 
tective substance  in  the  peritoneal  exudation.  Usually  this  fluid  was 
insufficient  to  protect  normal  guinea-pigs  against  typhoid  infection. 
In  a  few  experiments  only  was  the  exudation  found  to  be  as  pro- 
tective as  the  blood  serum  ;  in  most  of  the  others,  the  blood  serum 
was  much  more  active  than  the  fluid  of  the  exudation.  The  spleen 
was  the  organ  which  exhibited  the  greatest  protective  power,  and  [308] 
in  nearly  one  half  of  the  cases  it  was  more  active  than  was  the  blood. 
The  bone-marrow  sometimes  gave  analogous  though  much  less  marked 
results.  The  spleen  consequently  must  be  looked  upon  as  the  principal 
seat  of  the  production  of  the  protective  substance. 

Having  confirmed  this  observation  of  Pfeiffer  and  Marx  and  of 
Wassermann  and  Takaki,  Deutsch  tried  to  obtain  the  protective 
property  in  guinea-pigs  deprived  of  their  spleens.  The  experiment 
was  quite  successful,  and  here  again  his  result  agreed  with  that 
obtained  by  Pfeifter  and  Marx.  Guinea-pigs  from  which  the  spleen 
had  been  removed  developed  the  protective  property  just  as  well  as 
did  the  control  animals  ;  in  the  former  the  bone-marrow  was  found  to 
be  specially  active. 

When  Deutsch,  instead  of  removing  the  spleen  from  his  guinea- 
pigs  before  the  injection  of  the  micro-organisms,  did  so  some  (3 — 5) 
days  afterwards,  there  often  occurred  a  marked  diminution  in  the 
amount  of  the  protective  substance  produced.  We  must  conclude, 
therefore,  that  soon  after  inoculation  there  appeal's  in  the  spleen 
1  Ann.  de  Flnst.  Pasteur,  Paris,  1899,  t.  xin,  p.  689. 


294  Chapter  IX 

a  phenomenon  which  is  associated  with  the  development  of  the 
protective  power.  The  most  simple  explanation  of  these  facts  is 
that  the  micro-organisms  injected  into  the  peritoneal  cavity  and  soon 
afterwards  seized  by  the  phagocytes  (for  the  most  part  by  the  micro- 
phages),  are  carried  to  the  phagocytic  organs,  particularly  the  spleen, 
lymphatic  glands,  and  bone-marrow.  In  those  animals  whose  spleens 
are  left  intact  a  large  number  of  these  microphages  loaded  with 
micro-organisms  make  their  way  into  this  organ,  a  fact  confirmed  by 
direct  observation.  When  the  spleen  is  removed  the  microphages 
must  necessarily  betake  themselves  to  other  phagocytic  organs.  As 
the  micro-organisms  undergo  intracellular  digestion  in  the  phago- 
cytes, it  is  very  difficult,  if  not  impossible,  to  follow  them  for  any 
length  of  time  after  they  have  been  ingested,  but  the  analogy  with 
the  phenomena  of  the  resorption  of  red  blood  corpuscles,  described 
in  Chapter  IV,  justifies  us  in  concluding  that  in  the  case  of  micro- 
organisms matters  go  on  in  much  the  same  way.  These  organisms, 
seized  at  the  seat  of  inoculation  by  the  phagocytes,  are  transported 
by  these  cells,  in  their  peregrination  through  the  organs,  into  the 
general  circulation.  The  interpretation  I  have  just  given  has  been 
accepted  by  Deutsch. 

This  observer  wished  also  to  come  to  some  conclusion  as  to  the 
origin  of  the  agglutinative  property  so  well  developed  in  the  fluids 
of  animals  inoculated  with  the  typhoid  cocco-bacillus.  He  did  not 
[309]  succeed  in  solving  this  question,  but  he  was  able  to  demonstrate  the 
undoubted  difference  between  this  property  and  the  protective  power. 
The  facts  brought  forward  by  Deutsch  must,  therefore,  be  ranged 
alongside  the  many  others,  reported  on  above,  which  demonstrate  in 
the  most  conclusive  fashion  that  these  two  powers  of  the  body  fluids 
are  essentially  distinct. 

Such  concordant  results  obtained  by  all  investigators  who  have 
studied  the  origin  of  the  protective  power  warrant  the  conclusion 
that  it  is  the  elements  of  the  phagocytic  organs,  that  is  to  say,  the 
phagocytes  themselves,  which  produce  the  protective  substance.  But 
it  will  be  asked :  Can  we  therefore  accept  the  fixative  substance  or 
fixative  as  being  derived  from  the  same  source  ?  •  When  the  ex- 
periments I  have  just  summarised  were  carried  out  the  fixatives 
were  not  as  yet  sufficiently  known  and  were  confounded  with  the 
protective  substances.  Nevertheless,  there  can  be  no  doubt  as  to 
what  the  answer  to  the  question  just  put  must  be.  In  the  account 
of  the  experiments  of  Pfeiffer  and  Marx  we  find  very  precise  state- 


Acquired  immunity  against  micro-organisms      295 

merits  as  to  the  granular  transformation  of  the  vibrios.  Thus,  they 
observed  on  several  occasions  that  an  extract  of  the  spleen  set  up 
this  transformation  in  a  particularly  distinct  and  rapid  fashion  at 
a  period  when  the  blood  and  serum,  used  in  a  much  stronger  dose, 
were  incapable  of  producing  the  same  effect.  Xow,  as  Pfeiffer's 
phenomenon  is  a  visible  manifestation  of  the  action  of  the  specific 
fixative,  it  cannot  be  doubted  that  the  spleen  is  really  the  principal 
seat  of  development  of  the  fixative  substance  before  it  makes  its 
appearance  in  the  blood. 

Before  concluding  this  chapter  we  must  review  very  briefly  the 
principal  phenomena  associated  with  acquired  immunity  against 
micro-organisms.  The  extracellular  destruction  of  these  parasites 
takes  place  in  the  living  animal  under  special  conditions  only,  when 
the  phagocytes  suffer  a  temporary  injury  (phagolysis)  and  allow  their 
microcytases  to  escape.  These  latter  by  no  means  represent  attributes 
of  the  body  fluids,  as  is  even  yet  maintained  by  some  writers.  These 
soluble  ferments  are  connected  with  the  phagocytes  and  represent  the 
ferments  of  intracellular  digestion.  The  cytases  undergo  no  modifi- 
cation during  the  process  of  immunisation  and  correspond  to  those 
which  act  in  natural  immunity. 

The  agglutinative  substance  often  present  in  the  normal  fluids 
of  the  body  becomes  much  more  developed  in  those  of  immunised 
animals.   It  is  truly  humoral,  as  it  circulates  in  the  plasmas  and  passes 
into  the  fluid  exudations  and  transudations.    But  the  part  played  by  [310] 
it  in  immunity  is  very  restricted. 

The  protective  and  fixative  properties,  most  often  closely  con- 
nected with  each  other,  are  very  markedly  developed  in  an  animal 
enjoying  acquired  immunity.  They  may  act  upon  the  micro- 
organisms which  become  permeated  by  the  fixative  substance,  or 
upon  the  infected  animal  by  stimulating  its  defensive  reaction,  but 
they  are  incapable  of  affecting  the  vitality  or  virulence  of  the  micro- 
organism. The  two  properties  (protective  and  fixative)  reside  in  the 
fluids  of  the  body,  but  they  are  functions  of  the  cell  products.  The 
elements  of  the  phagocytic  organs  (spleen,  bone-marrow,  lymphatic 
glands),  or  phagocytes,  produce  the  specific  protective  and  fixative 
substances  which  pass  thence  into  the  plasmas. 

The  phagocytic  reaction  is  very  general  in  acquired  immunity. 
The  phagocytes  which  have  a  very  imperfect  antimicrobial  function 
or  none  at  all,  become,  as  the  result  of  vaccination,  much  more 
active.  They  exhibit  a  very  marked  positive  chemiotaxis  and 


296  Chapter  IX 

acquire  the  faculty  of  digesting  micro-organisms  in  a  greatly  intensi- 
fied degree.  It  is  with  the  increase  of  this  digestive  power  that  we 
have  connected  the  over-production  by  the  phagocytes  of  the  fixative 
and  protective  substances  which  are  excreted  in  large  quantities  by 
these  cells  and  pass  into  the  fluids  of  the  animal.  As  these  substances 
are  phagocytic  products  it  may  be  readily  conceived  that  in  certain 
examples  of  acquired  immunity  the  animal  overcomes  the  micro- 
organisms without  the  protective  substances  being  found  in  the  fluids. 
It  is  sufficient  that  it  is  in  the  possession  of  the  phagocytes,  which 
may  retain  it  within  themselves  and  not  throw  it  off*  into  the  circu- 
lation. 

From  this  account  it  will  be  seen  that  the  phenomena,  in  acquired 
immunity  against  micro-organisms,  are  merely  a  more  or  less  stereo- 
typed copy  of  those  that  are  presented  in  the  animal  after  the 
resorption  of  cells.  There,  also,  we  have  intracellular  digestion  with 
over-production  of  specific  fixatives,  part  of  which  are  excreted  and 
thus  pass  into  the  plasmas.  In  the  resorption  of  cells  there  is  also  a 
double  action  of  cytases  and  fixatives ;  but  in  this  case  the  macro- 
cytases  intervene,  whilst  in  the  resorption  of  micro-organisms  this 
function  is  performed  by  the  microcytases.  The  fixatives  in  the  two 
cases  are  very  different  from  the  point  of  view  of  their  action,  for 
[311]  they  are  specific  ;  but  the  cells  which  act  in  their  production  belong, 
in  both  cases  (resorption  of  animal  cells  and  of  micro-organisms),  to 
the  category  of  phagocytes. 

It  is  often  maintained  that  the  theory  I  have  just  summarised  is 
fundamentally  opposed  to  the  theory  of  side-chains  or  receptors 
formulated  by  Ehrlich1.  This  view  I  cannot  accept.  Applied  to 
acquired  immunity  against  micro-organisms  this  theory  may  be 
summed  up  as  follows.  The  micro-organisms,  when  inoculated  in  a 
non-lethal  but  immunising  dose,  combine  with  certain  cells  of  the 
animal.  The  receptors  of  the  micro-organisms  find  corresponding 
receptors  in  these  cells,  but,  when  once  combined,  the  receptors  of  the 
,  cells  become  incapable  of  fulfilling  their  normal  nutritive  function. 
The  cells,  thus  deprived  of  their  receptors,  reproduce  such  an 
enormous  quantity  of  them  that  a  portion  is  excreted  into  the 
surrounding  medium  and  passes  into  the  plasmas.  These  receptors, 
originating  from  cells,  but  which  have  become  constituent  parts  of 
the  body  fluids,  are  nothing  but  the  fixatives  or  intermediary  bodies, 

1  Ehrlich,  Lazarus  u.  Piukus,  "Leukaemie,  etc."  in  Nothnagel's  "Specielle  Pathologie 
u.  Therapie,"  Wien,  1901,  Bd.  vm,  I  Theil,  in  Heft,  Schlussbetrachtungen,  S.  163. 


Acquired  immunity  against  micro-organisms      297 

or  the  amboceptors  of  Ehrlich.  On  a  fresh  arrival  of  the  same 
micro-organisms,  they  meet  with,  in  the  fluid  of  the  exudations, 
numerous  amboceptors  which  combine  with  the  corresponding 
receptors  of  the  micro-organisms,  without,  however,  destroying  them 
or  interfering  with  their  vitality.  As  these  amboceptors  possess  still 
a  second  affinity,  that  for  the  molecules  of  the  cytases,  or  the 
"  complements "  of  Ehrlich,  the  micro-organisms  can  be  placed  in 
contact  with  these  soluble  ferments.  Without  the  intervention  of  the 
fixatives,  the  combination  of  the  body  of  micro-organisms  with  the 
cytase  can  never  take  place,  because  the  receptors  of  the  micro- 
organisms are  not  adapted  to  those  of  the  cytases.  When  the 
molecules  of  these  ferments  are  found  in  the  plasmas  in  a  free 
state,  they  can  be  attacked  by  the  corresponding  group  of  the 
amboceptors. 

Let  us  compare  the  theory  we  have  just  sketched  with  that 
described  further  back.  The  micro-organisms,  inoculated  with  a 
non-lethal  but  immunising  dose,  are,  as  we  have  seen,  ingested 
by  the  phagocytes  and  afterwards  digested  within  them.  This  intra- 
cellular  digestion  is  followed  by  the  over-production  of  the  specific 
fixative,  of  which  a  part  is  excreted  and  passes  into  the  plasmas. 
These  are  the  results  of  the  well-established  experimental  data  [312] 
described  in  this  chapter.  Ehrlich's  theory  is  in  no  way  in  oppo- 
sition to  this  ;  it  simply  attempts  to  penetrate  more  deeply  into  the 
mechanism  of  the  phenomena  observed  as  taking  place  between 
the  micro-organism  and  the  cell.  The  act  which  we  simply  term 
intracellular  digestion  is  divided  by  Ehrlich  into  its  constituent  parts. 
According  to  him  there  is  a  combination  of  the  fixative,  on  the  one 
hand,  with  the  molecule  of  the  micro-organism,  on  the  other,  with  that 
of  the  soluble  ferment  or  cytase.  According  to  Ehrlich  it  is  the 
amboceptors  of  the  cells  which  become  detached  in  order  to  furnish 
the  fixative  that  circulates  in  the  plasmas.  For  us  there  is  simply  an 
over-production  of  one  of  the  two  ferments  of  intracellular  digestion, 
without  defining  more  exactly  what  constituent  part  of  this  ferment 
passes  into  the  circulation.  The  two  theories  may  supplement  each 
other  but  are  in  no  way  contradictory  in  principle.  There  is  only  a 
single  important  point  wherein  they  do  not  accord.  Ehrlich  thinks 
that  the  cytases  are  always  free  in  the  body  fluids  and  that  the  cells, 
in  order  to  exert  a  digestive  action  on  the  micro-organisms,  must 
previously  seize  their  molecules  by  means  of  one  of  the  groups  of  their 
amboceptors.  We,  on  the  contrary,  have  developed  the  idea  that  the 


298  Chapter  IX 

cytases  are  only  free  in  the  animal  during  phagolysis  and  that  under 
normal  conditions  the  cytases  remain  closely  bound  up  with  the 
phagocytes.  This  statement  is  based  upon  a  large  number  of  well- 
established  experimental  facts  and  must  therefore  be  accepted  as 
proved.  It  does  not,  however,  affect  any  fundamental  principle  of 
Ehrlich's  theory.  On  the  other  hand  the  bases  of  Ehrlich's  theory 
aifect  none  of  the  main  features  of  the  theory  I  have  developed.  The 
doctrine  which  regards  acquired  immunity  as  a  particular  case  of 
resorption  may  be  reconciled  with  the  conception  of  amboceptors. 
But  it  accords  equally  well  with  Bordet's  conception,  according  to 
which  the  fixatives  act  not  as  intermediary  substances  between  the 
micro-organism  and  the  cytase,  but  as  substances  which  sensitise  the 
micro-organisms  for  the  penetration  of  the  digestive  ferment.  This 
delicate  question  has  not  yet  been  definitely  settled,  but  Bordet's 
experiments  described  in  Chapter  IV  are  greatly  in  favour  of  this 
view. 

Neisser  and  Wechsberg1  have  tried  to  obtain  some  idea  of  the 
[313]  manner  in  which  the  fixatives  act  on  the  micro-organisms  and  have 
recorded  a  series  of  very  interesting  facts.  They  have  shown  that 
these  substances  only  bring  about  the  destruction  of  bacteria  when 
they  are  in  certain  relations  with  the  cytase.  Mixtures  of  fixatives 
and  cytases  in  which  the  former  are  found  in  excess  not  only  do  not 
kill  the  micro-organisms  but  even  allow  them  to  develop  abundantly. 
To  attain  this  result  Neisser  and  Wechsberg  mixed  constant  quantities 
of  bacteria  and  normal  serum  containing  cytase  with  variable  quanti- 
ties of  the  serum  of  immunised  animals  heated  to  56°  C.  As  we 
know,  this  specific  serum,  as  the  result  of  being  thus  heated,  is 
deprived  of  its  cytases,  but  may  be  readily  made  active  again  by  the 
addition  of  normal,  unheated  serum.  This  paradoxical  fact,  demon- 
strated by  Neisser  and  Wechsberg  can,  in  their  opinion,  be  explained 
only  by  Ehrlich's  theory  of  amboceptors.  When  these  bodies  with 
double  affinities  are  found  in  too  large  quantity  as  regards  the  cytase, 
it  may  happen  that  one  part  only  of  those  which  combine  with  the 
receptors  of  the  micro-organisms  succeed  in  linking  to  themselves  the 
molecules  of  the  active  ferment.  The  amboceptor ,  being  by  itself 
incapable  of  destroying  the  micro-organism,  can  be  injurious  to  it 
only  on  condition  that  it  brings  cytase.  Consequently  as  the  amount 
of  this  cytase  is  too  small  for  the  much  larger  number  of  ambo- 
ceptors we  can  readily  conceive  that  the  micro-organisms  may  profit 
1  Munchen.  med.  Wchnschr.,  1901,  p.  697. 


Acquired  immunity  against  micro-organisms      299 

tli  ereby  and  remain  alive.  This  interpretation  is  certainly  very  ingenious, 
but  nothing  proves  that  it  corresponds  with  the  real  state  of  things. 
Neisser  and  Wechsberg  have  themselves  observed  that  the  serum 
of  the  normal  goat  can  also  prevent  the  bactericidal  action  of  the 
cytase.  In  this  case,  however,  they  suggest  the  intervention  of  an 
anticytase  of  this  normal  serum.  The  same  explanation  might 
perhaps  serve  also  to  explain  the  preventive  action  of  the  serum  of 
immunised  animals.  We  know  that  anticytases  are  found  frequently 
enough  in  the  various  serums  and  that  they  undergo  great  variations, 
according  to  the  conditions  present  in  the  animals  furnishing  the 
blood. 

In  any  case,  it  is  evident  that  the  theory  of  receptors  must  in 
no  way  be  regarded  as  the  antithesis  of  the  theory  of  phagocytosis. 
This  latter  quite  retains  its  right  to  affirm  that,  in  acquired  im- 
munity against  micro-organisms,  phagocytes  play  the  most  general 
and  important  part.  They  hold  back  the  cytases  which  are 
capable  of  ridding  the  animal  of  micro-organisms  from  destroying 
them.  It  is  further  these  same  cells  that  produce  and  excrete  the  [314] 
fixative  and  protective  substances.  The  free  fixatives  may  attack  the  . 
micro-organisms  in  the  body  fluids  but  they  are  incapable  of  depriv- 
ing them  of  life  or  even  of  virulence.  The  cytases,  after  escaping 
from  the  phagocytes,  may  certainly,  in  collaboration  with  the  fixatives, 
destroy  a  certain  number  of  the  micro-organisms,  but  only  in  special 
cases  met  with,  no  doubt,  but  only  rarely,  under  natural  conditions. 
On  the  other  hand,  the  phagocytes  in  the  animal  which  enjoys 
acquired  immunity  constantly  fulfil  the  function  of  seizing  the 
micro-organisms  and  of  submitting  them  in  their  interior  to  the 
combined  action  of  fixatives  and  cytases. 

Acquired  immunity,  like  natural  immunity  against  micro-organisms, 
presents  merely  special  phases  of  intracellular  digestion. 


CHAPTER    X 

[315]  RAPID  AND  TEMPORARY  IMMUNITY  AGAINST  MICRO- 
ORGANISMS, CONFERRED  BY  SPECIFIC  AND  NORMAL 
SERUMS,  OR  BY  OTHER  SUBSTANCES,  OR  BY  MICRO- 
ORGANISMS OTHER  THAN  THOSE  AGAINST  WHICH 
IT  IS  DESIRED  TO  PROTECT  AN  ANIMAL 

Immunity  conferred  by  specific  serums. — Analogy  of  the  mechanism  of  this  immunity 
with  that  observed  in  immunity  obtained  with  pathogenic  micro-organisms  and 
their  products. — Part  played  by  phagocytosis  in  the  immunity  conferred  by 
specific  serums. — Influence  of  opium  on  the  course  of  immunisation  by  these 
serums. — Stimulant  action  of  specific  serums. — Protective  and  stimulant  action 
of  normal  serums. — Influence  of  fluids,  other  than  serums :  broth,  urine,  physio- 
logical saline  solution,  etc. 

Antagonism  between  anthrax  and  certain  bacteria. 

WE  have  seen  how  important  in  the  study  of  acquired  immunity 
against  micro-organisms  is  the  demonstration  of  the  protective  power 
of  the  body  fluids.  Without  being  absolutely  general,  this  power  is, 
nevertheless,  widely  diffused  and  is  found  even  in  certain  examples  of 
acquired  immunity  against  micro-organisms  belonging  to  the  animal 
kingdom.  Up  to  the  present  I  have  refrained  from  doing  more  than 
point  out  the  presence,  in  the  fluids  of  the  immunised  animal,  of  this 
protective  property  and  have  studied  it  exclusively  in  relation  to  the 
animal  that  produces  it.  We  must  now  pass  to  the  question  :  How 
do  the  protective  substances  act  in  the  animal  which  receives  them 
ready  formed  ?  This  immunity,  which  has  received  from  Ehrlich  the 
name  of  passive  immunity  against  micro-organisms,  must  now  be 
examined. 

The  study  we  now  propose  to  enter  upon  is  rendered  much  easier 
from  our  study  of  the  data  acquired  on  the  phenomena  exhibited  in 
the  animal  vaccinated  with  micro-organisms  or  their  products,  data 
already  given  in  the  preceding  chapter.  There  is,  indeed,  a  very 
striking  analogy  between  the  two  kinds  of  immunity,  and  though  we 
draw  a  sharp  line  of  distinction  between  them,  this  is  due  to  the  fact 


Immunity  against  micro-organisms  301 

that  the  immunity  conferred  by  micro-organisms  or  their  products 
requires  some  time  for  its  development  and  endures  for  a  long  period,  [316] 
whilst  the  immunity  due  to  the  introduction  of  specific  serums  into  an 
animal  is  set  up  immediately,  but  endures  for  a  very  short  time  only. 

The  diseases  of  the  Invertebrata  being  seldom  due  to  the  micro- 
organisms that  produce  infections  in  the  higher  animals,  the  question 
as  to  whether  the  Invertebrata  can  be  immunised  by  means  of  pro- 
tective serums  has  not  yet  been  studied.  Still,  we  already  have  certain 
ideas  on  the  protection  of  lower  vertebrates  by  specific  serums. 
Gheorghiewsky1,  in  my  laboratory,  carried  out  some  experiments  on 
this  point.  He  found  that  the  serum  of  mammals  (guinea-pig,  goat) 
immunised  against  the  Bacillus  pyocyaneus,  was  under  certain  con- 
ditions capable  of  protecting  the  green  frog  against  a  dose  of  this 
organism  that  was  always  fatal  to  the  control  animals.  When 
injected  along  with  the  Bacillus  pyocyaneus,  the  serum  did  not 
prevent  a  fatal  infection  ;  sometimes  this  infection  developed  even 
more  rapidly  than  in  the  control  frogs.  It  was  only  when  the  pro- 
tective injection  was  made  24  or,  better  still,  48  hours  before  the 
inoculation  of  the  bacilli,  that  the  protective  action  became  evident. 
The  serum  used  in  these  experiments  was  not  bactericidal  for  the 
Bacillus  pyocyaneus  which  grew  most  luxuriantly ;  but  it  agglutinated 
a  large  proportion  of  the  bacilli.  Gheorghiewsky  pointed  out,  how- 
ever, that  frogs  injected  with  cultures  agglutinated  by  the  goat's  serum 
died  just  as  readily  as  did  the  control  animals.  As  the  phagocytic 
reaction  was  invariably  very  active  in  those  frogs  which  resisted  the 
virus,  after  the  injection  of  protective  serum,  it  is  very  probable  that 
this  fluid  exercises  a  stimulant  influence  on  the  phagocytes. 

This  idea  of  stimulation  by  anti-infective  serums  in  cases  of  tem- 
porary immunity  conferred  by  these  fluids,  has  already  been  set  forth 
in  my  researches  on  the  immunity  of  rabbits  against  the  Gentilly 
cocco-bacillus,  induced  by  the  serum  of  vaccinated  rabbits.  This 
view,  however,  has  not  been  favourably  received,  especially  in  view 
of  the  discovery  of  the  phenomenon  of  the  transformation  of  cholera 
vibrios  into  granules.  Pfeifler  himself  noted  that  this  transformation 
took  place  not  only  in  the  peritoneal  cavity  of  vaccinated  guinea-pigs 
but  also  in  the  peritoneal  cavity  of  normal  guinea-pigs,  into  which  he  [317] 
had  injected  small  quantities  of  specific  serum.  As  this  latter  fluid, 
in  Pfeiffer's  hands,  was  incapable  of  transforming  the  vibrios  into 
granules  in  vitro,  he  concluded  that  the  cellular  elements  of  the 
1  Ann.  de  VInst.  Pasteur,  Paris,  1899,  t.  xin,  p.  315. 


302  Chapter  X 

normal  animal  were  endowed  with  the  power  of  modifying  the  in- 
active substance  of  the  specific  serum  into  bactericidal  substance. 
According  to  this  conception  the  immunity  conferred  by  this  serum 
was  not  entirely  passive  since,  in  order  to  prepare  the  substance 
which  transforms  and  kills  the  vibrios,  the  co-operation  of  the  living 
cells  was  necessary. 

My  demonstration  of  the  possibility  of  obtaining  Pfeiffer's  pheno- 
menon in  vitro  at  once  turned  the  balance  in  favour  of  the  theory 
that  the  immunity  induced  by  the  specific  serum  is  due  to  a  direct 
humoral  action  upon  the  micro-organism.  Under  these  conditions 
such  immunity  could  only  be  interpreted  as  being  purely  passive. 
This  view  seemed  to  be  finally  established  by  Bordet's  discovery  that 
a  specific  serum,  inactive  by  itself,  became  capable  of  producing 
Pfeiffer's  phenomenon,  as  soon  as  a  small  quantity  of  normal,  non- 
specific serum  was  added  to  it.  Bordet1  thus  sums  up  his  theory  of 
the  immunity  conferred  by  specific  serums :  "  Passive  immunity  is 
due,  in  part  at  least,  to  a  chemical  action  exerted  on  the  vibrios  by 
two  pre-formed  substances,  the  one  present  in  the  animal  before  any 
injection  is  made,  the  other  found  in  the  serum  that  is  injected;  this 
phenomenon  is  purely  chemical  in  the  sense  that  it  can  go  on  without 
the  aid  of  a  vital  reaction,  of  any  new  cell  secretion :  indeed  it  is 
found  to  take  place  in  fluids  from  which  the  cells  have  been  entirely 
removed"  (p.  217).  These  demonstrations  led  up  to  the  belief  that 
the  organism  of  the  animal  remained  absolutely  passive  when  it  was 
subjected  to  the  action  of  protective  or  anti-infective  serums,  and  that 
the  case  of  the  cholera  vibrio  represented  a  kind  of  schema,  which 
was  applicable  to  the  whole  of  the  group  of  phenomena  met  with  in 
passive  immunity. 

As  in  the  study  of  the  immunity  obtained  as  the  result  of  vaccina- 
tions with  micro-organisms  or  their  products,  so  in  "passive  immunity" 
there  was  seen  only  the  direct  chemical  action  of  two  substances  on 
the  micro-organism,  and  efforts  were  made  to  extend  this  demonstration 
to  a  series  of  infective  diseases. 

[318]  Pfeiffer  and  Kolle2  having  observed  that  the  blood  serum  of 
persons  convalescent  from  typhoid  fever,  as  well  as  that  of  animals 
vaccinated  with  the  typhoid  bacillus,  exhibited  a  great  protective 
power  for  the  guinea-pig,  wished  to  get  some  idea  of  the  mechanism 
of  this  immunity.  They  found  that  in  the  peritoneal  cavity  of  guinea- 

1  Ann.  de  VInst.  Pasteur,  Paris,  1896,  t.  x,  p.  193. 
58  Ztschr.  f.  Hyg.,  Leipzig,  1896,  Bd.  xxi,  S.  203. 


Immunity  against  micro-organisms  303 

pigs,  inoculated  with  the  typhoid  cocco-bacillus  and  simultaneously 
subjected  to  the  action  of  protective  serums,  the  micro-organisms 
lose  their  mobility  almost  immediately.  A  little  later,  they  exhibit  a 
degeneration  of  form,  become  less  refractile  and  disintegrate.  After 
the  injection  of  large  doses  of  specific  serum  the  bacilli,  much  as  in 
the  case  of  the  cholera  vibrio,  become  transformed  into  granules. 
"  But,"  say  these  authors,  "  this  last  mode  of  destruction,  that  is  to 
say  the  formation  of  granules  at  the  expense  of  the  injected  bacteria, 
does  not  occur  with  such  remarkable  regularity  as  it  does  in  Pfeiffer's 
phenomenon  in  the  cholera  vibrio"  (p.  219).  Whilst  these  changes 
are  going  on  in  the  peritoneal  fluid,  the  leucocytes  begin  to  come  up 
and  to  ingest  the  bacilli  and  their  dtbris.  "Phagocytosis,  therefore, 
undoubtedly  plays  a  part  in  the  destruction  of  the  bacteria.  Never- 
theless, as  most  of  the  injected  bacteria  die  in  the  fluid  of  the 
exudation,  phagocytosis  can  not  be  regarded  as  the  cause  of  the 
protective  action  of  the  serum  "  (p.  220).  We  see  from  this  descrip- 
tion that  even  in  the  case  of  the  typhoid  cocco-bacillus  the  direct 
action  of  the  fluids  of  the  body  is  perceptibly  less  marked  than  in 
the  case  of  the  cholera  vibrio.  Even  in  the  latter,  however,  it  is 
necessary  to  make  many  reservations.  The  same  laws  apply  to  the 
immunity  against  this  micro-organism,  conferred  by  the  serum  of 
immunised  animals,  as  to  the  immunity  due  to  vaccinations  by  the 
vibrios  or  their  products.  To  treat  this  subject  fully  one  would  have 
to  repeat  almost  textually  the  two  preceding  chapters,  but  I  will 
simply  recall  the  fact  that  this  transformation,  almost  general  and 
very  rapid,  as  we  observed  in  vitro  in  vibrios  placed  in  contact  with 
fresh  specific  serum  or  with  the  mixture  of  this  serum,  heated  to 
65° — 56°  C.,  and  normal  unheated  serum,  is  only  met  with  in  the 
animal  body  where  phagolysis  appears.  Pfeiffer  first  observed 
the  phenomenon  which  bears  his  name  in  the  peritoneal  cavity, 
and  it  is  best  seen  in  that  situation  during  the  period  of  the 
phagolysis  of  the  white  corpuscles.  Vibrios,  mixed  with  small  doses  [319] 
of  specific  serum  which  by  itself  is  able  to  render  them  motionless 
and  agglutinate  them,  but  which  is  absolutely  unable  to  transform 
them  into  granules,  present  this  transformation  immediately  they  are 
introduced  into  the  peritoneal  cavity  of  normal  guinea-pigs.  In  this 
case  the  vibrios,  permeated  by  the  fixative  of  the  specific  serum,  are 
affected  by  the  microcytase  which  has  escaped  from  the  injured 
phagocytes  and  is  found  in  the  peritoneal  fluid.  The  preparation  of 
the  peritoneal  cavity  of  normal  guinea-pigs  by  means  of  an  injection 


304  Chapter  X 

of  broth  or  physiological  saline  solution  the  day  before,  prevents  the 
production  of  Pfeiffer's  phenomenon,  in  spite  of  the  protective  serum, 
just  as  in  vaccinated  guinea-pigs.  In  both  cases  the  vibrios,  without 
being  transformed  into  granules  by  the  fluid  part  of  the  peritoneal 
exudation,  are  ingested  by  the  phagocytes  en  masse  and  with  extra- 
ordinary rapidity.  This  experiment  was  repeated  by  Gamier1  with  the 
typhoid  cocco-bacillus.  He  first  injected  into  the  peritoneal  cavity 
of  young  guinea-pigs  several  c.c.  of  physiological  salt  solution,  of  fresh 
broth  or  of  some  other  fluid.  The  next  day  he  introduced  into  the 
same  situation  typhoid  cocco-bacilli  mixed  with  blood  serum  from 
a  donkey  that  had  been  for  a  long  time  immunised  against  this 
organism.  A  few  minutes  (2 — 4)  after  this  latter  injection  the  leuco- 
cytes, whose  phagolysis  had  been  prevented  by  the  previous  day's 
preparation,  were  found  crammed  with  cocco-bacilli.  Some  of  these 
bacilli,  like  those  still  free  in  the  peritoneal  fluid,  retained  their  normal 
form,  but  a  very  large  number  of  those  ingested  by  the  microphages 
were  already  transformed  into  granules.  This  experiment  affords 
fresh  confirmation  of  the  hypothesis  that  the  substance  which  trans- 
forms the  cocco-bacilli  or  the  vibrios  into  granules  is  the  microcytase. 
In  the  phagocytes  in  their  normal  condition  this  microcytase  is  found 
in  the  microphages,  but  during  phagolysis  a  portion  of  it  escapes  into 
the  surrounding  fluid.  In  the  control  experiments  made  by  Garnier 
with  young  normal  guinea-pigs  not  prepared  by  preliminary  injection, 
the  simultaneous  injection  of  typhoid  cocco-bacilli  and  specific  donkey's 
serum  set  up  this  attenuated  and  not  very  typical  Pfeiffer's  phenomenon 
described  in  Pfeiffer  and  Kolle's  memoir. 

Soon  after  the  discovery  of  Pfeiffer's  phenomenon  I2  was  able 
to  bring  forward  a  proof  that  it  was  produced  neither  in  the  sub- 
[320]  cutaneous  tissue,  in  the  oedemas  set  up  by  the  arrest  of  the  circulation, 
nor  in  the  anterior  chamber  of  the  eye  of  animals  when  cholera  vibrios 
mixed  with  anti-infective  specific  serum  were  injected  into  these 
situations.  Here  the  micro-organisms  retain  their  normal  form, 
remain  quite  alive  and  in  this  condition  are  ingested  by  the  leuco- 
cytes which  are  brought  up  to  the  points  invaded.  These  cells, 
attracted  by  the  vibrionic  products,  do  not  undergo  any  phagolysis 
and,  untrammelled,  fulfil  their  phagocytic  function.  •  Inside  them  are 
found  vibrios  which  have  kept  their  elongated  form  and  others  which 
have  become  transformed  into  granules.  The  exudations  containing 

1  Ann,  de  VInst.  Pasteur,  Paris,  1897,  t.  xi,  p.  773. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  453. 


Immunity  against  micro-organisms  305 

these  elements  still  give  cholera  cultures  on  nutrient  media,  a  proof 
that  some  at  least  of  the  intracellular  vibrios  are  alive.  Here  we 
have  no  destruction  of  the  micro-organisms  in  the  fluids  of  the 
body,  consequently  no  direct  action  of  the  bactericidal  substance. 
This  substance,  enclosed  in  the  phagocytes,  can  only  act  through  the 
intervention  of  these  elements. 

Mesnil1  made  analogous  experiments  with  the  Massowah  vibrio, 
which,  unlike  the  cholera  vibrio,  is  peculiarly  virulent  when  injected 
subcutaneously  into  guinea-pigs.  In  spite  of  this  difference,  this 
micro-organism,  when  injected  along  with  protective  serum  into 
normal  animals,  behaves  much  as  does  the  cholera  vibrio  proper. 
Mesnil  injected  the  Massowah  vibrios  at  the  same  time  as  the  anti- 
infective  specific  serum,  into  the  subcutaneous  tissue  of  young  and 
adult  guinea-pigs  and  of  young  rabbits.  In  every  case  he  observed  the 
same  reaction  phenomena  in  the  animal  organism.  The  vibrios  caused 
the  formation  of  oedema  at  the  point  of  inoculation  and  remained 
isolated  in  the  fluid.  The  majority  of  these  micro-organisms  became 
motionless,  but  a  few  remained  motile.  Pfeiffer's  phenomenon  was 
never  observed.  There  was  sometimes  an  aggregation  of  the  vibrios, 
but  this  was  not  comparable  with  the  marked  agglutination  brought 
about  by  the  specific  serum  in  vitro.  The  vibrios  retained  their  power 
of  reproduction,  and  Mesnil  was  able  to  observe  them  in  all  phases  of 
division.  Some  hours  (6—8)  after  inoculation  the  leucocytes  began 
to  come  up  to  the  seat  of  injection  and  set  to  work  at  once  to  ingest 
the  vibrios.  This  phagocytosis  became  more  and  more  marked,  and 
finally  there  was  ingestion  of  the  Avhole  of  the  micro-organisms.  Drops 
of  the  exudation  containing  only  intraphagocytic  vibrios,  when  placed  [321] 
in  the  incubator,  gave  abundant  cultures.  The  leucocytes  died  out- 
side the  animal  body,  whilst  the  vibrios  continued  to  live  and  grow 
well  under  the  new  conditions.  Certain  leucocytes  became  three  times 
their  original  size,  and  their  contents  were  seen  to  be  made  up  of 
vibrios  closely  packed  together.  The  subcutaneous  exudation,  when 
withdrawn  even  eight  days  after  the  injection  of  the  micro-organisms 
and  sown  on  nutrient  media,  still  gave  colonies  of  vibrios. 

It  is  evident,  therefore,  that  the  direct  action  of  the  protective 
serum  on  the  vibrios  was  reduced  to  a  mere  trifle.  It  rendered  them 
motionless  and  brought  about  a  very  slight  clumping,  but  it  was 
incapable  of  transforming  the  vibrios  into  granules  or  of  destroying 
them.  We  see,  then,  that  even  in  the  case  of  the  vibrios,  the  part 

1  Ann.  de  VInst.  Pasteur,  Paris,  1896,  t.  x,  p.  371. 
B.  20 


306  Chapter  X 

played  by  Pfeiffer's  phenomenon  is  very  limited.  The  destruction 
of  the  vibrios  is  effected  with  certainty,  and  completely,  under  the 
influence  of  the  specific  serums,  not  by  a  direct  action  of  the  two  anti- 
bacterial substances  but  through  the  mediation  of  the  phagocytes. 
Before  the  fixative,  introduced  with  the  protective  serum,  can  bring 
about  this  result,  the  leucocytes,  impressed  with  a  special  sensitive- 
ness, must  come  up  to  the  seat  of  inoculation,  seize  the  micro-organisms 
and  secrete  around  them  their  cytase.  It  is  only  as  a  result  of  these 
actions,  purely  vital,  that  the  chemical  or  physico-chemical  reaction  of 
the  substances  which  intervene  in  the  destruction  of  the  vibrios  is 
brought  about. 

Under  these  conditions  it  can  easily  be  understood  that  if  the 
vital  action  of  the  phagocytes  is  retarded  or  depressed  the  injection 
of  protective  serum  cannot  preserve  the  life  of  the  animal.  Canta- 
cuzene1,  who  had  already  made  a  similar  demonstration  on  guinea-pigs 
vaccinated  against  the  cholera  vibrio  by  these  organisms  or  by  their 
products,  carried  out  numerous  experiments  on  the  action  of  opium 
on  normal  guinea-pigs  simultaneously  inoculated  with  vibrios  and 
specific  serum.  Before  injecting  this  mixture  Cantacuzene  narcotised 
his  animals  by  means  of  tincture  of  opium.  The  great  majority  (f) 
of  the  guinea-pigs  so  treated  died  at  the  end  of  one  or  several  days. 
The  transformation  of  the  vibrios  into  granules,  under  the  influence 
of  the  serum,  took  place  in  the  peritoneal  cavity,  but  the  leucocytes, 
on  account  of  the  narcotic  action  of  the  opium,  were  tardy  in  coming 
up.  On  their  arrival  in  the  peritoneal  cavity  they  were  capable  of 
[322]  ingesting  the  granules,  but  absolutely  refused  to  seize  entire  vibrios, 
always  fairly  numerous  in  the  exudations.  In  spite  of  the  appearance 
of  a  large  number  of  leucocytes,  these  cells  were  still  too  weak  to 
offer  any  adequate  opposition  to  the  vibrios,  which  increased  in 
number  and  continued  to  multiply  up  to  the  death  of  the  animal, 
when  the  exudation  simply  swarmed  with  very  motile  vibrios.  Some- 
times the  struggle  was  prolonged.  The  weakened  leucocytes  allow 
the  vibrios  to  develop,  but,  after  a  greater  or  less  length  of  time, 
they  regain  their  strength  and  begin  to  ingest  the  micro-organisms 
vigorously.  Complete  phagolysis  follows,  but  the  guinea-pig,  attacked 
by  the  toxic  products  of  the  vibrio,  finally  succumbs  in  spite  of  the 
absence  of  free  vibrios  from  its  body. 

An  analysis  of  the  phenomena  observed  in  the  body  of  an  animal 
treated  with  antivibrionic  serum,  demonstrates  that,  in  spite  of  a 
1  Ann.  de  Vlnst.  Pasteur,  Paris,  1898,  t.  xn,  p.  290. 


Immunity  against  micro-organisms  307 

certain  direct  action  of  the  substances  contained  in  this  fluid,  there 
still  remain  a  whole  series  of  processes,  amongst  which  the  carriers  of 
the  cytases,  that  is  to  say  the  phagocytes,  fill  the  most  important  role. 
Nevertheless,  the  cholera  vibrio  with  its  allied  forms  is  still  the  most 
sensitive  of  all  the  micro-organisms  to  the  bactericidal  action  of  the 
fluids  of  the  body.  It  may,  therefore,  readily  be  conceived  that  the 
more  resistant  micro-organisms  are  even  less  subject  to  the  direct 
influence  of  the  specific  serums.  Thus  we  have  seen  that  the  cocco- 
bacillus  of  typhoid  fever  presents,  in  the  phagolysed  peritoneal  fluid, 
merely  an  attenuated  form  of  Pfeiffer's  phenomenon.  The  other 
representatives  of  the  group  of  bacilli  are  still  less  subject  to  the 
direct  action  of  the  serums,  and  Gheorghiewsky1,  in  his  studies  on 
the  Bacillus  pyocyamm,  found  that  normal  guinea-pigs,  injected 
subcutaneously  with  anti-infective  specific  serum,  and  inoculated  into 
the  peritoneal  cavity  with  this  organism,  present  the  same  phenomena 
as  those  described  in  Chapter  VIII.  He  never  noticed  either  lysis 
of  the  bacteria  in  the  fluids  of  the  animal  or  their  total  transforma- 
tion into  agglutinated  masses  outside  the  phagocytes.  The  resistance 
offered  by  the  animal  was  always  in  direct  relation  to  the  rapidity  of 
the  appearance  and  extent  of  the  phagocytic  reaction. 

In  order  to  determine  the  relative  importance  of  each  of  the  factors 
which  act  in  the  preservation  of  animals  subjected  to  the  influence 
of  the  specific  serum,  Gheorghiewsky  repeated  Cantacuzene's  experi-  [323] 
ments  on  the  effect  of  narcotisation  by  tincture  of  opium.  This  alkaloid 
retards  diapedesis,  but  does  not  affect  the  tactile  sensibility  or  the 
motility  of  the  leucocytes.  The  humoral  properties,  on  the  other 
hand,  are  not  in  the  least  affected  by  the  narcosis.  In  spite  of  the 
fact  that  in  guinea-pigs,  narcotised  and  treated  with  anti-infective 
serum,  the  direct  action  was  not  interfered  with,  the  animals  always 
died  because  the  retarded  and  incomplete  phagocytic  reaction  was 
insufficient  to  destroy  the  bacilli  rapidly  enough. 

Mesnil2  studied  the  action  of  the  specific  serum  against  swine 
erysipelas  on  normal  animals  into  which  he  had  injected  it  some  time 
before  inoculation  of  the  corresponding  bacillus  into  the  peritoneal 
cavity.  This  serum  exercises  a  most  marked  protective  action  on  the 
mouse,  an  animal  very  susceptible  to  the  pathogenic  action  of  this 
micro-organism.  In  mice  so  prepared  complete  and  rapid  phago 
cytosis  takes  place.  These  micro-organisms  before  being  ingested 

1  Ann,  de  FInst.  Pasteur,  Paris,  1899,  t.  xni,  p.  312. 
8  Ann.  de  llnst.  Pasteur,  Paris,  1898,  t.  xn,  p.  492. 

20—2 


308  Chapter  X 

by  the  phagocytes  show  no  appreciable  change;  they  are  always 
stained  very  uniformly  and  intensely  by  Gram's  method,  and  they 
never  swell  up.  The  bacilli  undergo  no  agglutination  in  the  body 
of  the  mouse,  a  fact  of  which  we  can  convince  ourselves  by  examining 
hanging  drops  of  the  exudation.  The  phenomenon  which  strikes  the 
observer  most  is  the  very  pronounced  phagocytosis,  due  principally 
to  the  activity  of  the  microphages.  Some  hours  after  inoculation 
these  cells  are  found  to  be  crammed  with  bacilli,  a  large  number 
of  which  no  longer  stain  in  the  normal  fashion.  Without  being  trans- 
formed into  granules,  these  micro-organisms  undergo  intracellular 
digestion  which  at  the  end  of  a  few  days  is  complete.  This  de- 
struction is  more  rapid  and  complete  in  the  microphages,  slower  in 
the  macrophages.  Drops  of  exudation  collected  from  these  mice,  at 
a  stage  when  the  ingestiou  is  completed,  produce  fatal  septicaemia 
in  untreated  mice.  This  is  proof  that  at  the  moment  when  they  were 
seized  by  the  phagocytes  the  bacilli  still  retained  their  virulence. 
Mesnil,  as  the  result  of  his  experiments,  concludes  that  "the  effect 
of  the  serum  is  to  stimulate  the  phagocytes  and  especially  the  poly- 
nuclear  forms;  they  ingest  more  quickly,  they  digest  more  quickly. 
The  serum  is,  therefore,  a  stimulant  of  the  cells  charged  with  the 
defence  of  the  animal "  (p.  496). 

[324]  We  need  not  describe  the  phenomena  produced  in  mice  inoculated 
subcutaneously  and  treated  with  protective  serum,  for  even  in  the 
peritoneal  cavity  neither  Pfeiffer's  phenomenon  nor  any  extracellular 
destruction  of  the  bacilli  can  be  observed.  The  micro-organisms, 
when  subjected  to  the  influence  of  the  specific  serum,  readily  absorb 
the  fixative,  as  demonstrated  by  Bordet  and  Gengou1.  This  absorp- 
tion must  certainly  favour  the  action  of  the  intraphagocytic  cytases. 
It  is  not,  however,  sufficient  to  explain  the  protective,  anti-infective 
action  of  the  serum.  Such  explanation  was  given  by  the  experiments 
which  Gengou,  at  my  request,  was  good  enough  to  make.  He  inocu- 
lated mice  with  the  bacilli  of  swine  erysipelas,  mixed  with  specific 
serum  heated  to  55°  C.,  to  which  was  added  some  normal  guinea-pig's 
serum.  The  mice  so  treated  resisted  the  infection  but  controls  died 
in  a  few  days.  Being  thus  assured  of  the  protective  action  of  the 
serum,  Gengou  prepared  the  same  mixtures  of  swine  erysipelas 
bacilli  and  of  the  two  serums  ;  but,  instead  of  injecting  the  whole  of 
the  mixture,  he  removed  the  bacilli  from  the  serums,  after  a  pro- 
longed contact,  and  injected  the  bacilli  alone  into  the  mice.  The 
1  Ann.  de  FInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  289. 


Immunity  against  micro-organisms  309 

bacilli  had  become  permeated  with  fixatives,  but,  in  spite  of  this, 
they  killed  the  mice  almost  as  quickly  as  the  controls.  Consequently, 
it  is  not  the  fixative  adherent  to  the  micro-organisms  which  deter- 
mines the  protective  action  of  the  specific  serum.  This  fluid  must 
contain  another  substance,  one  that  will  stimulate  the  phagocytes. 

The  analysis  of  the  mechanism  of  the  immunity  termed  passive,  that 
is  to  say;  communicated  to  normal  animals  by  the  introduction  of  an  anti- 
infective  specific  serum,  teaches  us  that,  even  when  the  direct  action  of 
the  humoral  substances  is  very  limited,  the  protective  effect,  thanks  to 
the  stimulant  action  which  brings  about  the  destruction  of  the  micro- 
organisms through  the  mediation  of  the  phagocytic  reaction,  is  still 
produced.  The  result  at  which  we  have  thus  arrived  is  confirmed  by 
the  examination  of  the  phenomena  observed  in  animals  subjected  to 
the  action  of  anti-anthrax  serum.  Marchoux1  first  supplied  us  with 
precise  details  as  to  the  mode  of  action  on  the  rabbit  of  the  serum  of 
animals  treated  with  anthrax  bacilli.  He  found  that,  in  the  peritoneal 
cavity  of  rabbits  injected  the  day  before  with  anti-anthrax  serum,  the 
inoculated  anthrax  bacilli  almost  immediately  become  the  prey  of  [325] 
phagocytes.  Within  a  couple  of  minutes  after  the  introduction  of 
bacilli  into  the  peritoneal  cavity,  the  great  majority  of  them  are 
ingested  by  the  leucocytes  ;  ten  minutes  later,  there  are  no  free 
bacilli.  Not  only  the  ingestion  but  also  the  destruction  of  these 
micro-organisms  takes  place  with  great  rapidity,  and  even  a  few  hours 
after  the  injection,  the  peritoneal  exudation,  when  sown  on  nutrient 
media,  remains  sterile.  In  the  subcutaneous  tissue  the  phagocytic 
reaction  requires  a  longer  time  than  in  the  peritoneal  cavity,  never- 
theless, it  goes  on  very  rapidly.  Thus,  when  inoculated  into  the 
subcutaneous  tissue  of  the  ear  of  rabbits  treated  with  specific  serum, 
the  bacilli  are  in  great  part  ingested  at  the  end  of  half-aii-hour.  At 
the  end  of  an  hour  phagocytosis  is  usually  complete. 

In  March oux's  experiments  the  importance  of  the  part  played  by 
the  phagocytes  becomes  still  more  prominent  when  it  impedes  their 
function  in  any  way.  Rabbits  injected  with  anti-anthrax  blood  and 
24  hours  later  inoculated  below  the  skin  of  the  ear  with  anthrax 
bacilli  always  resist  infection,  exhibiting  the  well-marked  phago- 
cytosis just  mentioned.  In  other  rabbits,  however,  prepared  in  the 
same  way  with  the  serum,  but  inoculated  the  following  day  into  an 
ecchymosis  excited  by  tapping  the  ear  lightly,  a  certain  number  of 
the  bacilli  escape  the  phagocytes  and  succeed  in  setting  up  an 
1  Ann.  de  Flnst.  Pasteur,  Paris,  1895,  t  ix,  p.  800. 


310  Chapter  X 

abundant  oedema  followed  by  a  fatal  anthrax  at  the  end  of  a  few  days. 
On  making  a  post-mortem  examination  of  these  animals  the  bacilli 
were  not  numerous,  but  they  were  found  in  all  the  organs.  The  same 
result  was  obtained  in  another  experiment  in  which  Marchoux  inocu- 
lated subcutaneously  with  anthrax  blood  which  coagulated  in  situ 
rabbits  prepared  with  specific  serum.  The  blood  clot  attracted  only 
the  macrophages,  as  pointed  out  in  Chapter  IV.  The  microphages 
did  not  come  up  until  late  and  then  in  small  numbers.  Now,  as 
these  are  the  phagocytes  that  are  chiefly  instrumental  in  destroying 
the  anthrax  bacillus,  their  absence  allowed  the  bacilli  to  multiply  and 
to  set  up  a  fatal  anthrax.  The  rabbits  prepared  with  the  same  serum 
but  injected  with  anthrax  blood  diluted  with  broth  (which  prevents 
the  formation  of  clot)  completely  resisted  infection,  thanks  to  the 
phagocytic  reaction  which  went  on  without  hindrance. 

Sclavo1  also,  who  made  numerous  investigations  on  the  action 
[326]  of  the  anti-anthrax  serum,  is  of  opinion  that  this  action  is  not  a 
direct  one  upon  the  bacillus  but  is  produced  indirectly  through 
the  action  of  the  animal  organism.  He  maintains  that  the  serum 
stimulates  the  function  of  the  phagocytes  and  augments  the  bacteri- 
cidal action  of  the  body  fluids.  But  since  this  bactericidal  power 
enters  the  cytase  as  a  substance  destroying  the  micro-organisms,  and 
this  cytase  is  contained  in  the  phagocytes,  we  can  readily  understand 
what  a  dominant  part  in  the  process  these  elements  play. 

Sobernheim2,  also,  has  paid  much  attention  to  the  question  now 
under  discussion.  As  the  result  of  his  researches  he  comes  to  the 
conclusion  that  the  anti-anthrax  serum  "  cannot  exert  any  effect  on 
the  virus  by  a  direct  action  of  the  protective  specific  substances."  In 
order  that  the  serum  may  be  effective,  the  active  intervention  of  the 
organism  of  the  animal  is  necessary,  otherwise,  it  is  impossible  to 
explain  why  the  serum,  used  in  the  same  proportion  against  the  same 
quantity  of  anthrax  bacilli,  should  protect  one  species  of  animals 
(the  rabbit)  and  allow  another  (guinea-pig,  mouse)  to  succumb. 
When  Sobernheim  tried  to  apply  to  anthrax  the  discovery  of  the 
transformation  of  cholera  vibrios  into  granules,  he  got  only  negative 
results.  There  was  nothing  produced  comparable  to  Pfeiffer's  phe- 
nomenon and  the  anthrax  bacilli  usually  underwent  no  apparent 
modification.  Sobernheim  affirms  also  that  the  rapid  phagocytosis 
under  the  influence  of  the  serum,  described  by  Marchoux,  "  does  not 

1  Centralbl.f.  Bakteriol  u.  Parasitenk.,  Jena,  1899, 1  Abt.,  Bd.  xxvi,  S.  428. 

2  Ztschr.f.  Hyg.,  Leipzig,  1899,  Bd.  xxxi,  S.  110. 


Immunity  against  micro-organisms  311 

appear  to  be  produced  under  all  circumstances"  (p.  117).  As,  how- 
ever, his  researches  on  this  subject  were  made  on  guinea-pigs  which, 
in  spite  of  the  treatment  with  specific  serum,  always  ended  by 
succumbing  to  anthrax,  we  readily  understand  that  his  results  cannot 
be  compared  with  those  obtained  by  Marchoux.  I  was  present  at  the 
experiments  of  this  observer  and  convinced  myself  of  the  accuracy  of 
the  facts  recorded  in  his  memoir. 

Most  of  the  examples  here  studied  justify  fully  the  hypothesis 
of  the  stimulant  action  of  protective  serums,  a  view  that  I  formu- 
lated as  the  result  of  my  researches  on  the  immunity  of  rabbits 
against  the  Gentilly  cocco-bacillus1.  In  this  the  first  case  of  anti- 
infective  immunity,  due  to  the  serum  elaborated  by  an  immunised 
animal,  I  could  not  find  either  a  bactericidal  action,  however  slight, 
or  any  agglutinative  or  attenuating  property  of  the  fluids  of  the 
body.  As,  on  the  other  hand,  this  serum  had  no  antitoxic  power,  [327] 
everything  indicated  that  we  must  look  for  its  action,  which  was 
nil  or  very  slight  on  the  micro-organism,  as  being  exerted  on  the 
organism  of  the  animal  into  which  it  was  injected  for  protective 
purposes.  A  comparative  examination  of  the  course  of  the  pheno- 
mena in  the  subcutaneous  tissue  of  the  ear  in  rabbits,  some  of 
which  received  an  injection  of  the  specific  serum  into  the  veins  whilst 
others  were  kept  as  controls,  at  once  showed  how  widely  different 
were  the  two  cases.  In  the  control  animals,  the  cocco-bacilli  im- 
mediately began  to  multiply  without  meeting  with  any  opposition  on 
the  part  of  the  organism  of  the  animal ;  on  the  other  hand,  in  the 
rabbits  treated  with  serum,  the  serum  became  rich  in  leucocytes 
which  at  once  set  to  work  to  ingest  the  micro-organisms.  In  course 
of  time  the  latter  gradually  diminished  in  numbers,  whilst  the  leuco- 
cytes went  on  increasing.  The  phagocytosis,  also,  became  more  and 
more  marked.  This  struggle  was  continued  for  more  than  24  hours, 
after  which  the  purulent  exudation,  containing  masses  of  leucocytes, 
no  longer  included  any  cocco-bacilli  visible  under  the  microscope 
either  outside  or  inside  cells.  Nevertheless,  this  pus  was  still  capable 
of  producing  a  fatal  septicaemia  in  untreated  rabbits,  clearly  proving 
that  it  still  contained  some  living  and  virulent  micro-organisms. 
These  cocco-bacilli  persisted  for  a  long  time  inside  the  phagocytes ; 
their  presence  being  demonstrated  by  injecting  the  exudation  into 
unprotected  rabbits  and  thus  setting  up  a  fatal  infection.  Finally, 
however,  they  disappear  completely.  On  consideration  of  such  facts 
1  Ann.de  VInst.  Pasteur,  Paris,  1892,  t.  vi,  p.  308. 


312  Chapter  X 

as  these  I  considered  that  I  was  justified  in  formulating  the  following 
conclusion  at  the  end  of  my  memoir  :  "  From  the  facts  I  have  de- 
scribed, taken  collectively,  we  may  draw  the  conclusion  that  the 
preservation  of  unvaccinated  rabbits  treated  with  serum  is  due  to  a 
superactivity  of  the  phagocytic  defence ;  and  it  is  allowable  to 
express  the  opinion  that  the  protective  serum  of  hog  cholera  acts 
in  rabbits  by  stimulating  the  phagocytes,  rendering  them  less  sensi- 
tive to  the  toxins,  and  by  stimulating  them  in  their  struggle  against 
the  bacteria"  (p.  310).  The  facts  since  collected  by  various  observers 
fully  justify  this  hypothesis.  Amongst  the  other  micro-organisms 
against  which  a  rapid  immunisation  has  been  obtained  by  means 
of  serum,  we  must  cite  the  cocco-bacillus  of  bubonic  plague. 
Numerous  experiments,  carried  out  on  several  species  of  animals, 
have  shown  that  antiplague  serum  markedly  augments  the  phago- 
cytic reaction. 

[328]  In  the  group  of  the  cocci,  the  streptococci  have  been  specially 
fully  studied  from  the  point  of  view  now  under  discussion.  As 
already  stated  in  another  chapter,  success  has  been  attained  not  only 
in  thoroughly  immunising  several  species  of  animals  against  this 
dreaded  micro-organism  but  active  serums  have  been  obtained 
capable  of  conferring  distinct  and  certain  immunity.  The  protective 
action  of  Marmorek's  serum,  prepared  at  the  Pasteur  Institute,  has 
been  specially  carefully  studied.  This  serum  is  obtained  from  horses 
that  have  received  numerous  injections  of  various'  races  of  strepto- 
cocci pathogenic  for  animals  and  for  man1.  At  Lou  vain,  Denys  and 
his  pupils  prepared  several  other  antistreptococcic  serums  and  studied 
their  protective  effect  on  laboratory  animals. 

In  collaboration  with  Leclef,  Denys2  began  by  vaccinating  rabbits 
against  streptococci  and  studied  the  mechanism  of  the  immunity 
obtained  in  these  animals.  A  summary  of  their  researches  will  be 
found  in  the  eighth  chapter.  Denys  and  Leclef  considered  that  the 
serum  of  vaccinated  rabbits  intervenes  in  two  ways,  first  by  directly 
hindering  the  multiplication  of  the  streptococcus  and  then  by  exalting 
the  activity  of  the  leucocytes.  They  applied  these  results  to  the  case 
in  which  immunity  is  conferred  upon  normal  rabbits  by  the  inter- 
vention of  the  serum  of  the  vaccinated  rabbit,  but  they  were  unable 
to  furnish  any  data  bearing  directly  on  this  immunity.  Somewhat 

1  Marmorek,  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  593. 

2  La  Cellule,  Lierre  et  Louvain,  1895,  t.  xi,  p.  175,  and  Bull.  Acid.  roy.  de  med. 
de  Belg.,  Bruxelles,  1S95. 


Immunity  against  micro-organisms  313 

later,  Denys1,  in  collaboration  with  Marchand,  published  another 
memoir  in  which  he  describes  experiments  on  the  mechanism  of  the 
immunity  conferred  on  rabbits  by  injections  of  the  blood-serum  of 
vaccinated  horses.  From  these  experiments  they  draw  the  conclusion 
that  "the  serum  of  the  horse  immunised  against  the  streptococcus 
possesses  no  bactericidal  properties,  properly  so  called,  against  this 
micro-organism ;  it  does  not  affect  it  directly ;  but  it  contains  a 
substance  which  renders  the  phagocytic  power  of  the  leucocytes 
extremely  active.  Even  in  the  presence  of  small  quantities  of  this 
serum,  the  white  corpuscles  rapidly  ingest  the  streptococci  and  are 
capable  of  stopping  all  development  so  long  as  they  retain  their 
amoeboid  movements."  "The  action  of  the  serum  upon  the  leuco- 
cyte in  its  conflict  with  the  streptococcus,  is  really  derived  from  the 
horse  immunised  against  this  organism.  It  exists  neither  in  the  [329] 
ordinary  horse  nor  in  the  horse  vaccinated  against  diphtheria" 
(p.  15).  Against  these  experiments  of  Denys  and  Marchand  we 
might  bring  the  same  objection  that  we  raised  against  the  analogous 
experiments  of  Denys  and  Leclef,  because,  in  both  cases,  these 
writers  lay  too  much  stress  on  the  presence  or  absence  of  the  phe- 
nomena of  phagocytosis  in  preparations  kept  outside  the  body  of  the 
animal.  Under  these  conditions  phagocytosis  is  effected  in  a  fashion 
too  artificial  to  be  capable  of  furnishing  exact  information. 

Von  Lingelsheim2  met  Denys  and  Marchand  with  the  fact  that, 
in  their  researches,  the  serum  of  the  horse  immunised  against 
the  streptococcus  was  only  feebly  bactericidal.  After  a  prolonged 
contact  (6 — 12  hours)  with  a  specific  serum,  the  streptococci,  when 
transferred  to  rabbit's  blood,  showed  retarded  development  as  com- 
pared with  streptococci  subjected  to  the  influence  of  the  anti- 
diphtheritic  and  antitetanic  horse  serum.  Von  Lingelsheim  himself, 
however,  points  out  that  the  bactericidal  action  of  the  antistrepto- 
coccic  serum  was  feeble  and  transient,  and  required  the  intervention 
of  the  reaction  of  the  animal  cells  within  the  body. 

The  researches  carried  out  by  Bordet3  in  my  laboratory  are  not 
open  to  the  objections  that  we  were  justified  in  putting  forward 
against  the  experiments  made  by  Denys  and  Marchand,  since  he  care- 
fully watched  the  phenomena  of  immunity  as  they  developed  in  the 

1  Bull  Acad.  roy.  de  med.  de  Belg.,  Bruxelles,  1896. 

2  Habilitations-Schrift,   Marburg,  1899,  and  in  von  Behriug's  "Beitrage  zur 
experimentellen  Therapie,"  1899,  Bd.  r,  S.  40. 

3  Ann.  de  I'Inst.  Pasteur,  Paris,  1897,  t.  xi,  p.  177. 


314  Chapter  X 

body  of  the  animal  subjected  to  the  action  of  antistreptococcic  horse 
serum.  Bordet  began  by  studying  the  properties  of  this  serum  and 
accepted  Denys'  and  Marchand's  statement  that  bactericidal  power, 
however  small,  was  absent.  The  streptococcus  grows  as  well  in 
this  serum  as  it  does  in  that  of  the  untreated  horse.  In  the  specific 
serum,  however,  markedly  longer  chains  are  produced  than  in  normal 
serum.  This  difference  is  found  only  in  the  earliest  period  of  the 
growth.  The  agglutinative  power  of  the  antistreptococcic  serum  is 
but  feebly  marked.  The  injection  of  a  large  quantity  of  this  serum 
into  a  normal  rabbit  confers  no  bactericidal  power  upon  the  serum  of 
this  animal.  "  The  serum  extracted  24  hours  after  injection  is  quite 
as  suitable  for  use  as  a  culture  medium  as  that  furnished  by  the 
blood  before  the  introduction  of  the  serum.  In  both  the  micro- 
organism grows  rapidly  and  vigorously"  (p.  195).  Consequently,  in 
[330]  the  antistreptococcic  serum  there  is  nothing  comparable  to  what  we 
obtain  so  readily  with  antivibrionic  serum :  nothing  which  recalls 
Pfeiffer's  phenomenon,  even  of  an  attenuated  nature.  We  have 
already  noted  the  result  obtained  by  Bordet,  according  to  which  the 
streptococci,  developed  in  the  specific  horse  serum,  were  found  to  be 
endowed  with  their  normal  high  virulence. 

The  antistreptococcic  serum,  injected  into  the  peritoneal  cavity  of 
the  rabbit  the  day  previous  to  the  microbial  inoculation,  protects 
the  animal  absolutely,  provided  that  the  micro-organisms  be  not  too 
numerous  or  the  quantity  of  serum  not  too  small.  Under  these 
conditions  the  virus  is  ingested  pretty  rapidly  and,  so  far  as  we  know 
at  present,  completely.  The  micro-organism  is  thus  prevented  from 
developing  and  the  animal  remains  in  good  health,  whilst  the  control 
animal,  which  has  received  no  serum,  dies  in  a  very  short  time. 

When  the  number  of  streptococci  is  increased  the  effort  of  the 
animal  organism  to  get  rid  of  them  becomes,  in  spite  of  the 
protective  serum,  more  severe  and  much  more  prolonged.  Some 
of  the  micro-organisms  certainly  become  the  prey  of  phagocytes,  but 
a  sufficient  number  remain  free  in  the  peritoneal  cavity  to  multiply 
rapidly.  When  the  number  of  streptococci  has  become  sufficiently 
great  a  phenomenon,  to  which  Bordet  gives  the  name  of  "phagocytic 
crisis,"  is  suddenly  observed.  In  the  peritoneal  exudation,  which  has 
become  thick  and  has  taken  on  the  aspect  of  a  homogeneous  and 
white  pus,  a  most  rapid  phagocytosis  is  evidently  set  up.  In  a  short 
time  the  whole  of  the  streptococci,  which  were  swarming  outside  the 
cells,  are  ingested  by  the  leucocytes.  "The  essential  condition  for 


Immunity  against  micro-organisms  315 

recovery  is  always  this  complete  ingestion  "  (p.  203).  If  the  ingestion 
is  not  general,  the  rabbit  may  die,  although  much  later  than  the 
control  animal. 

The  phases  of  the  struggle  between  the  animal  organism,  when 
subjected  to  the  influence  of  the  protective  serum,  and  the  strepto- 
coccus, recall  Salimbeni's  experiments  on  immunised  horses.  The 
rabbit,  in  which  phagocytosis  could  not  take  place  at  once  owing  to 
the  presence  of  too  large  a  number  of  micro-organisms,  exhibits  first 
a  stage  of  free  development  of  the  streptococci,  after  which  the 
phagocytes  begin  to  fulfil  their  antibacterial  function.  Here  it  is 
especially  the  macrophages  which  act ;  the  microphages,  although 
present  in  fairly  large  numbers,  are  entirely  inactive.  This  first  stage 
of  phagocytic  reaction  is  insufficient.  It  is  followed  by  a  period  when  [331 
the  streptococcus  appears  to  gain  the  upper  hand.  Many  small 
chains,  having  escaped  the  phagocytes,  multiply  and  give  birth  to 
quite  a  new  generation  of  micro-organisms.  If  a  fresh  impulse  to 
phagocytosis  does  not  take  place  the  animal  dies  from  infection. 
When,  however,  the  protective  serum  has  been  of  sufficient  strength, 
a  new  army  of  leucocytes  arrives  on  the  scene  and  these  become 
masters  of  the  situation.  Phagocytosis  becomes  complete  and  micro- 
phages as  well  as  macrophages  devour  a  large  number  of  streptococci. 

Bordet,  who,  through  his  previous  investigations,  was  well  ac- 
quainted with  the  direct  action  of  the  protective  serum  on  vibrios, 
could  find  nothing  resembling  it  taking  any  part  in  the  struggle  of  the 
organism  of  the  animal  treated  with  antistreptococcic  serum  against 
the  streptococcus.  The  most  that  he  could  find  was  that  the  strepto- 
cocci which  again  begin  to  swarm  in  the  exudation  are  smaller  in  size 
than  the  normal  streptococcus.  It  must  be  accepted,  as  indicated  by 
the  most  recent  researches,  that  this  micro-organism  becomes  per- 
meated by  the  fixative  substance  of  the  specific  serum.  We  know 
already,  however,  that  this  fixation  cannot  deprive  the  micro-organisms 
of  their  virulence.  In  any  case,  then,  a  large  share  in  the  process 
must  be  attributed  to  the  action  of  the  phagocytes,  stimulated  by 
the  protective  serum,  in  the  struggle  of  the  animal  against  the 
streptococcus. 

Having  considered  this  series  of  examples  of  immunity  against 
bacteria  conferred  by  specific  serums,  we  are  in  a  position  to  form 
some  idea  of  the  mechanism  of  this  immunity.  Before  we  come  to  any 
general  conclusion,  it  may  be  useful  to  glance  at  an  example  of  this 
so-called  passive  immunity  against  a  micro-organism  belonging  to 


316  Chapter  X 

the  animal  kingdom.  Such  examples  are  not  numerous,  as,  in  the 
majority  of  cases  of  acquired  immunity  against  Protozoan  parasites, 
the  serum  is  inactive  and  incapable  of  communicating  immunity  to 
normal  individuals.  We  have  only  a  single  example,  the  Trypano- 
soma  of  rats,  against  which  Dr  Lydia  Rabinowitch  and  Dr  Kempner1 
have  demonstrated  the  possibility  of  immunisation  by  the  blood  serum 
of  vaccinated  white  rats.  The  mechanism  of  this  immunity  has  been 
studied  by  Laveran  and  Mesnil2,  who  found  that  it  was  like  that 
described  (Chap.  VIII)  in  connection  with  the  immunity  in  white  rats, 
conferred  by  the  inoculation  of  living  Trypanosomata.  The  specific 
[332]  serum  does  not  affect  these  infusoria  except  that  it  brings  about  slight 
agglutination.  Trypanosomata  placed  in  contact  with  it  retain  their 
pristine  vitality  and  motility.  This  fact  led  Mme  Rabinowitch  and 
Dr  Kempner  to  advance  the  hypothesis  that  the  protective  action  of 
the  serum  must  depend  upon  its  antitoxic  power.  Since,  however,  in 
the  infection  of  rats  by  the  Trypanosomata,  the  toxic  action  is  very 
feeble  if  not  nil,  it  is  very  difficult  to  accept  this  view.  It  certainly 
appears  to  be  much  more  probable  that  the  serum  acts  in  this  case,  as 
in  many  others,  by  stimulating  the  phagocytic  reaction.  The  rapidity 
with  which  the  living  Trypanosomata  are  ingested  by  the  phagocytes 
has  been  shown  by  Laveran  and  Mesnil. 

Reviewing  the  whole  of  the  data  on  immunity  produced  under  the 
influence  of  anti-infective  or  protective  serums,  it  is  evident  that  they 
fall  under  two  main  categories.  On  the  one  hand  there  is  a  direct 
action  of  these  serums  on  the  micro-organisms,  an  action  that  is  either 
microbicidal  properly  so  called,  agglutinative,  or  fixative.  On  the 
other  hand,  a  stimulation  of  the  phagocytic  defence  which  leads  to 
the  final  destruction  of  the  micro-organisms  is  set  up.  This  last  factor 
is  general ;  even  in  the  case  where  the  direct  action  is  most  marked 
(vibrios  in  the  phagolysed  peritoneal  cavity),  its  importance  is  con- 
siderable. The  micro-organisms  which  can  be  deeply  injured  by  the 
direct  action  of  the  specific  serum  are  few  in  number.  In  most  cases 
this  action  is  a  feeble  one  and  needs,  for  its  completion,  effective 
co-operation  on  the  part  of  the  phagocytes.  In  this  respect  micro- 
organisms present  a  whole  gamut  which  begins  with  the  cholera 
vibrio,  the  micro-organism  most  sensitive  to  the  action  of  the  body 
fluids,  and  ends  with  the  Trypanosoma  of  the  rat,  a  flagellated 

1  Ztschr.f.  Hyg.,  Leipzig,  1899,  Bd.  xxx,  S.  251. 

8  Laveran,  "litres  et  travaux  scientifiques,"  Paris,  1901,  p.  39.  Ann.  de  Vlnst. 
Pasteur,  Paris,  1901,  t.  xv,  p.  673. 


Immunity  against  micro-organisms  317 

Infusorian  which  cannot  have  even  its  motility  affected  by  the  direct 
action  of  the  fluid  elements.  In  all  these  cases,  of  course,  the  im- 
munity conferred  by  the  serums  is  due  to  the  final  destruction  of 
the  micro-organisms  which  invariably  resolves  itself  into  the  same 
fundamental  act — digestion  by  the  cytases,  a  phenomenon  which  can 
only  be  produced  at  all  quickly  by  the  action  of  cytases  contained 
in  the  protective  serums  or  that  have  escaped  from  the  phagocytes 
during  phagolysis.  The  digestion  by  the  cytases  may  also,  and  this 
is  usually  the  case,  be  effected  only  after  the  manifestation  of  a  regular 
series  of  vital  phenomena  on  the  part  of  the  defensive  elements 
of  the  body.  As  this  factor  fills  such  an  important  role,  it  is  readily 
understood  that  we  can  not  accept  the  term  passive  immunity  by 
which  to  designate  the  immunity  conferred  by  the  specific  serums. 
The  action  of  the  cytases,  which  is  necessary  to  bring  about  the  final  [333] 
result  in  this  immunity,  depends  too  much  on  the  activity  of  the 
cells  which  contain  the  bactericidal  ferment.  For  this  reason,  when 
the  functional  activity  of  the  phagocytes  is  in  abeyance  or  is  retarded, 
the  animal  succumbs,  in  spite  of  the  presence  in  its  organism  of  a 
more  than  sufficient  quantity  of  cytases.  In  this  connection  Wasser- 
mann's1  suggestion  of  adding  normal  serums  rich  in  cytases  to  the 
specific  serums  must  be  regarded  as  very  apposite.  When  protective 
serums  poor  in  cytases  or  which  have  lost  them  as  the  result  of 
heating,  of  the  use  of  antiseptics,  or  simply  from  the  influence  of 
time,  are  injected,  no  immunising  effect  is  ever  obtained,  simply  be- 
cause of  the  inactivity  of  the  phagocytes,  the  cells  in  which  the  cytases 
are  found.  If  at  the  same  time  normal  serum  rich  in  cytases  ready 
prepared  be  injected,  better  results  should  be  obtained.  We  may 
recall  here  an  analogous  example — the  anthrax  of  the  rat  Although 
possessing  a  large  quantity  of  cytase,  very  effective  against  the  bacillus, 
the  organism  of  the  rat  can  make  no  use  of  it,  because  the  phagocytes 
which  contain  it  do  not  manifest  a  sufficient  activity.  But  the 
injection  into  a  rat  of  blood  serum  from  the  same  species  containing 
a  certain  amount  of  cytase  that  has  escaped  during  the  formation  of 
the  clot,  is  sufficient  to  preserve  the  animal  against  a  fatal  infection. 

To  support  his  view,  sound  in  principle,  Wassermann  made  an 
experiment  the  interpretation  of  which  presents  certain  difficulties. 
He  injected  guinea-pigs  with  protective  antityphoid  serum,  in  a  dose 
insufficient  to  protect  them  against  a  fatal  infection.  By  introducing 
along  with  this  serum  a  certain  quantity  of  normal  ox  serum  which, 
1  Deutsche  med,  Wchnschr.,  Leipzig,  1900,  S.  285. 


318  Chapter  X 

by  itself,  is  also  incapable  of  averting  a  fatal  issue,  Wassermann 
obtains  an  absolute  immunity  of  his  animals.  This  immunity  is  due, 
according  to  Wassermann,  to  the  cytase  of  the  ox  serum  acting  along 
with  the  fixative  of  the  specific  serum.  The  united  action  of  the  two 
ferments  causes  the  death  of  the  micro-organisms.  Besredka1  has 
justly  observed  that  normal  ox  serum  contains,  in  addition  to  cytases, 
a  substance  which  exerts  a  distinct  agglutinative  action  on  the  typhoid 
cocco-bacillus  and  another  which  stimulates  the  phagocytic  action. 
These  two  substances  resist  a  temperature  of  55° — 60°  C.,  and  Besredka 
[334]  shows  that  with  normal  ox  serum,  deprived  of  its  cytases  by  heating 
as  above,  we  can  obtain  the  same  protective  effect  as  with  the  same 
serum  unheated. 

As  the  result  of  another  series  of  experiments,  Wassermann2 
recognises  the  immunising  action  of  normal  serum  heated  to  60°  C. 
and  so  entirely  deprived  of  its  cytases.  Into  the  peritoneal  cavity  of 
guinea-pigs  he  injects,  mixed  with  heated  normal  rabbit's  serum,  a 
dose  of  typhoid  cocco-bacilli  several  times  greater  than  the  lethal  dose. 
The  guinea-pigs  resist  this  completely.  Analysing  the  mechanism  of 
this  immunity,  Besredka  (l.c.  p.  229)  attributes  it  to  the  combined 
action  of  the  agglutinin  and  of  the  substance  which  stimulates  the 
phagocytes.  We  have  here  another  proof  that  the  stimulins  which 
play  such  an  important  part  in  immunity  conferred  by  serums,  are 
found  not  only  in  the  specific  serums,  but  also  in  normal  serums, 
whether  unheated  or  heated  to  55°— 60°  C. 

The  protective  property  of  the  normal  serums  of  man  arid  animals 
against  the  cholera  vibrio  has  already  been  referred  to.  We  may  now 
go  a  little  more  deeply  into  the  mechanism  by  which  these  serums 
act.  This  task  is  an  easy  one  thanks  to  the  important  work  by  Issaeff3 
carried  out  in  R.  Pfeiffer's  laboratory.  Having  confirmed  the  ob- 
servation, made  by  other  investigators,  that  blood  serum  from  the 
human  subject,  whether  in  health  or  affected  by  any  disease,  is  capable 
of  protecting  the  guinea-pig  against  the  cholera  vibrio  provided  that 
it  is  injected  24  hours  before  the  micro-organisms,  Issaeff  studied  the 
phenomena  observed  in  the  peritoneal  cavity  of  the  animals  experi- 
mented upon.  By  means  of  small  capillary  pipettes,  he  drew  off  at 
intervals  a  small  quantity  of  fluid  from  the  peritoneal  cavity  and 
examined  it  in  hanging  drop  or  in  stained  preparations.  Some  time 

1  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  225. 

2  Deutsche  med.  Wchnschr.,  1901,  S.  4. 

3  Ztschr.f.  Hyg.,  Leipzig,  1894,  Bd.  xvr,  S.  287. 


Immunity  against  micro-organisms  319 

after  the  injection  this  fluid  became  more  and  more  rich  in  leucocytes 
which  seized  the  vibrios,  ingested  and  destroyed  them.  To  obtain 
this  protective  effect  it  was  necessary  to  inject  from  O'l  to  5  c.c.  of 
human  blood  serum.  With  these  doses  he  could  prevent,  not  only 
infection  of  the  guinea-pigs  by  the  cholera  vibrio,  but  also  the  lethal 
effects  of  other  vibrios.  The  protective  action  of  normal  human  serum 
is  general,  therefore,  and  not  specific,  such  as  is  the  immunity  conferred 
by  the  serums  of  vaccinated  animals  or  of  the  human  subject  who  has 
suffered  from  an  attack  of  cholera. 

Shortly  afterwards  Funck1  confirmed  this  result  in  the  case  of  the  [335] 
typhoid  cocco-bacillus.  He  observed  that  normal  horse's  serum,  in- 
jected as  a  protective  agent  in  the  dose  of  half  a  c.c.  into  the  peritoneal 
cavity  of  the  guinea-pig,  preserved  this  animal  from  a  fatal  infection. 
Pfeiffer  and  Kolle  and  Chantemesse  and  Widal  obtained  the  same 
results  with  human  serum.  The  former  observers  lay  special  stress 
on  the  non-specific  character  of  this  protective  action  of  normal 
serums.  As  to  its  mechanism,  Funck  sums  it  up  as  follows :  "  the 
specific  serum  brings  about  a  rapid  lysis  of  the  bacilli,  normal  serum 
acts  in  a  much  more  limited  fashion;  if  the  dose  is  very  large  and 
if  the  animal  resists  infection,  the  phenomena  of  extracellular  de- 
generation are  rarely  appreciable,  and  it  seems  that  here  the  specially 
important  factor  is  the  intracellular  destruction  of  the  bacteria,  in  the 
phagocytes  "  (p.  70). 

Wassermann  has  shown  the  protective  action  of  normal  serum 
against  the  experimental  disease  produced  by  the  staphylococcus. 
This  action,  although  not  absolutely  general,  is  nevertheless  widely 
distributed.  Wassermann2,  from  comparative  investigations  on  this 
subject,  came  to  the  conclusion  that  "the  serum  of  a  different  species 
of  animal  acts  by  greatly  increasing  the  resistance,  whilst  the  serum 
of  the  same  species  produces  an  effect  which  is  not  nearly  so  marked." 
As  in  these  normal  serums  a  stimulating  influence  on  the  phagocytes 
is  specially  marked,  it  may  readily  be  understood  that  the  serum  of 
the  same  animal  or  of  the  same  species  does  not  produce  so  energetic 
an  effect  as  the  serum  of  a  different  species.  As  these  normal  serums 
possess,  not  only  the  property  of  exciting  phagocytosis,  but  often  also 
that  of  rendering  motionless  and  of  agglutinating  certain  micro-organ- 
isms, there  might  be  some  difficulty  in  interpreting  the  part  played 

1  "La  Serotherapie  de  la  fi&vre  typhoide,"  Bruxelles,  1896,  p.  69. 

2  Ztschr.f.  Hyg.,  Leipzig,  1901,  Bd.  xxxvn,  8.  199. 


320  Chapter  X 

by  these  serums.  It  may  be  useful,  therefore,  to  pass  in  review  the 
protective  action  of  fluids  less  complicated  than  blood  serums. 

Issaeff;  in  the  work  already  cited,  demonstrated  that  not  only 
normal  serums  but  a  whole  series  of  fluids,  such  as  urine,  broth,  etc., 
exert  a  protective  effect  against  microbial  infections.  These  fluids 
must  be  injected  about  24  hours  before  the  introduction  of  the 
bacteria.  The  best  method  consists  in  injecting  them  directly  into 
[336]  the  peritoneal  cavity,  after  which  the  animals  acquire  an  immunity 
against  absolutely  fatal  doses  of  cholera  vibrios.  Funck  verified  this 
observation  for  the  infection  caused  by  the  typhoid  cocco-bacillus, 
and  Bordet  confirmed  it  for  the  streptococcus.  The  injection  of 
peptonised  broth  into  the  peritoneal  cavity  of  the  normal  guinea-pig, 
made  24  hours  before  an  inoculation  of  double  the  fatal  dose  of  the 
streptococcus,  exerts  a  distinct  protective  action ;  the  infection  does  not 
kill  the  animal.  This  broth  is  neither  bactericidal,  attenuating,  nor 
agglutinative ;  it  forms  a  good  culture  medium  for  the  streptococcus 
and  possesses  no  fixative  power.  Consequently  it  does  not  act  directly 
on  the  vitality  or  virulence  of  the  micro-organism ;  nevertheless,  it  is 
distinctly  protective. 

According  to  Issaeff's  researches,  the  protective  substances  used 
by  him  must  be  arranged  in  the  following  order  as  regards  their 
action  against  the  cholera  vibrio.  Tuberculin  is  the  most  effective ; 
then  comes  a  2%  solution  of  nuclein,  followed  by  normal  human 
serum,  broth,  and  urine,  whilst  physiological  saline  solution  is  the 
least  active.  All  prevent  infection  by  the  vibrios,  but  the  protection 
is  effective  for  some  days  only;  this  protective  action  is  exerted 
against  various  kinds  of  bacteria,  being  in  no  sense  specific. 

Pfeiffer  lays  so  much  stress  on  the  great  difference  between  the 
protective  power  of  normal  serums,  as  well  as  of  the  other  fluids 
mentioned,  and  that  of  the  anti-infective  specific  serums,  that  he  even 
proposes  to  classify  the  first  group  as  giving  rise  to  pseudo-immunity 
or  resistance.  This  view  is  certainly  an  exaggerated  one,  because  it 
is  difficult  to  draw  a  very  distinct  line  between  the  two  groups  of 
phenomena.  There  are  normal  serums,  of  which  0*1  c.c.  is  quite 
sufficient  to  confer  the  protective  effect,  just  as  there  are  specific 
serums  of  which  it  is  necessary  to  make  use  of  a  much  greater  dose 
to  attain  the  same  result. 

Protective  fluids,  other  than  the  serums,  only  manifest  their  in- 
fluence by  exciting  a  great  phagocytic  "superactivity."  As  the  result 
of  their  injection  into  the  peritoneal  cavity  of  normal  guinea-pigs, 


Immunity  against  micro-organisms 


321 


first  a  transitory  phagolysis  is  induced,  this  being  soon  replaced  by  a 
very  considerable  afflux  of  leucocytes,  which  is  maintained  for  24  hours 
or  longer,  and  then  gives  place  to  the  normal  condition.    It  is  during 
the  period  of  the  greatest  leucocytosis 
of  the  peritoneal  fluid  that  the  animal 
exhibits  the  most  marked  resistance 
against    infective    micro-organisms. 
The    vibrios    are    rapidly    ingested 
by  the  phagocytes,  without  having 
previously  been  acted  upon  by  the 
"  humours."    Bordet  shows  that  the 
same  thing  happens  in  the  case  of 
the    streptococcus    inoculated   into 
guinea-pigs  after  a  protective  injec- 
tion of  peptonised  broth. 

We  have  observed  the  same  phe- 
nomenon in  guinea-pigs  and  white 
rats  inoculated  with  the  cocco- 
bacillus  of  plague.  Treated  with 
freshly  prepared  peptonised  broth  macrophage  from  guinea-pig. 


[337] 


FIG.  43.  Macrophage 
from  guinea-pig 
filled  -with  plague 
bacilli. 


FIG.  44.  Macrophage 
from  guinea-pig 
containing  plague 
bacilli  which  are 
commencing  to  es- 
cape from  the  pro- 
toplasm. 


FIG.  45.  Macrophage  from  guinea- 
pig  which  has  burst  as  the  result 
of  the  development  of  plague  bacilli 
within  it. 


the  day  previous  to  inoculation,  these  animals  oppose  to  the  micro- 
organism a  much  more  marked  resistance  than  do  the  control  animals. 
The  injection  of  the  cocco-bacillus  of  plague  sets  up  a  marked  phago- 
B.  21 


322  Chapter  X 

cytosis  on  the  part  of  the  macrophages.  These  cells  ingest  large 
numbers  of  micro-organisms  which,  after  a  time,  have  all  passed  into 
the  phagocytes.  If  a  drop  of  the  peritoneal  exudation  is  now  with- 
[338]  drawn,  we  find  only  intracellular  cocco-bacilli  (fig.  43).  If  the  drop  be 
kept  for  some  time  outside  the  animal  and  at  a  suitable  temperature 
the  macrophages  may  be  seen  to  perish  and  the  micro-organisms  to 
develop  in  their  contents.  We  thus  obtain  abundant  cultures  which 
pass  from  the  interior  of  the  macrophages  into  the  fluid  of  the 
exudation  (figs.  42,  44,  45).  When  the  animals  are  not  sufficiently 
protected  the  same  phenomenon  is  observed  in  the  peritoneal  cavity 
of  the  living  animal.  The  macrophages,  crammed  with  cocco-bacilli, 
burst,  allowing  the  micro-organisms  to  escape.  These  multiply  in  the 
peritoneal  fluid  and  spread  through  the  animal,  which  soon  dies. 

Wassermann  affirms  that  "the  artificially  increased  resistance  is 
nothing  but  an  active  and  reinforced  afflux  of  the  complements 
(cytases)  towards  one  point  in  the  animal,  for  the  purpose  of  di- 
gestion." (Ztschr.  f.  Hyg.,  Leipzig,  1901,  Bd.  xxxvn,  S.  199.) 
Wassermann  does  not  explain  how  this  afflux  of  cytases  is  produced. 
The  entirely  concordant  researches  on  this  point  by  Issaeff,  Funck, 
Bordet,  and  ourselves,  prove  that  this  afflux  takes  place  not  through 
the  mediation  of  the  fluids,  but  solely  through  the  phagocytes, 
the  carriers  of  the  cytases.  Consequently  it  is  beyond  dispute  that 
in  the  immunity  conferred  by  physiological  saline  solution,  broth,  and 
several  other  fluids,  we  have  to  do  solely  with  an  augmentation  of  the 
phagocytic  reaction.  In  the  immunity  conferred  by  normal  or  specific 
serums,  this  same  stimulating  factor  still  plays  the  more  important 
part.  Along  with  it,  however,  there  is  an  intervention  more  or  less 
pronounced,  according  to  circumstance,  and  more  or  less  frequent,  of 
cytases,  brought  by  the  serums  prepared  outside  the  body  or  that 
have  escaped  during  phagolysis,  as  well  as  of  substances  truly 
humoral,  such  as  the  fixatives  or  the  agglutinins. 

Amongst  the  non-specific  substances  which  are  capable  of  con- 
ferring an  immunity  more  or  less  stable,  must  be  placed  the 
products  of  micro-organisms  other  than  those  against  which  we  wish 
to  protect  the  animal.  Pasteur1  noted  that  when  the  anthrax 
bacillus,  mixed  with  other  micro-organisms,  in  themselves  inoffensive, 
is  inoculated  into  animals,  anthrax  does  not  develop  and  the  animals 
remain  well.  Later,  Emmerich2  showed  that  the  streptococcus  of 

1  Compt.  rend.  Acad.  d.  sc.,  Paris,  1877,  t.  LXXXV,  p.  107. 

2  Arch.f.  Hyg.,  Miinchen  u.  Leipzig,  1887,  Bd.  vi,  S.  442. 


Immunity  against  micro-organisms  323 

erysipelas   exerts    an    antagonistic    influence    against   the   anthrax 
bacillus.    He  succeeded  in  immunising  and  even  in  curing  rabbits  [339] 
inoculated  with  anthrax,  by  submitting  them  to  the  action  of  this 
streptococcus. 

These  experiments  served  as  the  starting-point  for  several  works 
on  the  vaccination  of  animals  against  anthrax  by  means  of  various 
micro-organisms,  as  well  as  by  their  products.  Pawlowsky1,  Watson- 
Cheyne2,  and  Bouchard3  have  proved  that  bacteria  not  very  patho- 
genic and  even  saprophytes,  such  as  the  Cocco-bacillus  prodigiosus, 
Friedlander's  bacillus,  and  the  Bacillus  pyocyaneus,  were  also  capable 
of  preventing  infection  by  the  anthrax  bacillus.  Freudenreich4 
showed  that  not  only  did  the  bacillus  of  blue  pus  exert  an  antago- 
nistic action  but  that  the  same  effect  could  be  obtained  with  sterilised 
cultures  of  this  organism.  Woodhead  and  Cartwright  Wood5  studied 
the  vaccinating  action  of  these  products  on  rabbits  inoculated  with 
virulent  anthrax  bacilli.  The  animals  resisted  completely  or  survived 
for  some  time.  Analysing  the  phenomena  produced  under  such  con- 
ditions, these  two  authors  came  to  the  conclusion  that  the  action  of 
sterilised  cultures  of  Bacillus  pyocyaneus  is  "  indirect  and  as  taking 
place  either  by  opposing  itself  to  the  action  of  the  poison  upon  the 
tissues,  or  by  stimulating  certain  tissues  and  increasing  their  func- 
tional activity."  With  the  object  of  obtaining  an  exact  interpretation 
of  this  antagonistic  influence  I  suggested  to  M.  Blagovestcheusky6 
that  he  should  investigate  in  detail  the  phenomena  which  take  place 
in  the  organism  of  rabbits  inoculated  with  the  anthrax  bacillus  and 
submitted  to  the  action  of  sterilised  cultures  of  the  Bacillus  pyo- 
cyaneus. At  the  very  outset  this  observer  was  met  by  the  fact  that 
these  cultures  act  directly  upon  the  vitality  of  the  anthrax  bacillus. 
Thus  the  association  of  the  former  with  the  anthrax  bacillus  in  vitro 
was  sufficient  to  interfere  with  the  development  of  the  latter.  Under 
these  conditions  he  had  to  renounce  the  investigation  of  the  part 
played  by  the  cellular  elements  of  the  rabbit  in  the  antagonism  of  the 
two  bacteria. 

Friedlander's  bacillus  has  been  found  to  be  much  more  suitable 
for  this  line  of  research  as  is  shown  by  work  carried  out  by  Freiherr 

1  Virchow's  Archiv,  Berlin,  1887,  Bd.  cvm,  S.  494. 

2  London  Medical  Record,  1887. 

3  Compt.  rend.  Acad.  d.  sc.,  Paris,  1889,  t.  cvm,  p.  713. 

4  Ann.  d.  Microgr.,  Paris,  1889,  p.  465. 

5  Compt.  rend.  Acad.  d.  sc.,  Paris,  1889,  t  cix,  p.  985. 

6  Ann.  de  FInst.  Pasteur,  Paris,  1890,  t.  iv,  p.  689. 

21—2 


324  Chapter  X 

[340]  von  Dungern1  in  my  laboratory.  This  observer  convinced  himself 
that  "  anthrax  bacilli  are  weakened  neither  by  the  encapsuled  bacilli 
nor  by  the  substances  which  they  contain."  These  micro-organisms 
do  not  interfere  in  the  slightest  with  the  anthrax  bacilli  either  outside 
or  within  the  animal,  and  "  when  the  anthrax  infection  does  not  be- 
come generalised  it  is  due  to  the  fact  that  the  anthrax  bacilli  are 
ingested  by  the  phagocytes  at  the  seat  of  inoculation  and  destroyed 
within  these  cells"  (p.  183). 

In  this  action  of  foreign  micro-organisms  upon  micro-organisms 
against  which  we  wish  to  protect  the  animal  we  have  to  deal  with 
something  analogous  to  the  condition  we  obtain  when  immunising 
with  normal  serums  or  with  any  other  kind  of  fluid.  In  both  cases 
immunity  is  rapidly  established,  but  it  is  very  transient  and  is  con- 
fined to  a  stimulation  of  the  phagocytic  resistance.  Direct  action 
may  also  intervene,  as  in  the  case  of  Bacillus  pyocyaneus,  but  it  is 
not  indispensable.  The  animal  whose  phagocytes  are  in  a  condition 
of  superactivity  can  do  without  this  direct  action,  its  own  resources 
being  sufficient  to  arrest  anthrax. 

Following  the  same  lines  of  investigation  as  those  on  the  an- 
tagonism between  the  anthrax  bacillus  and  several  other  micro- 
organisms, Klein2  has  demonstrated  that,  in  order  to  prevent  a 
guinea-pig  from  contracting  experimental  cholera  peritonitis,  it  is 
only  necessary  to  inject  into  it,  the  day  before  infection,  a  culture  of 
Tinkler  and  Prior's  vibrio  or  of  certain  other  bacteria.  These  ex- 
periments by  Klein  served  as  the  point  of  departure  for  Issaeff's  work 
which  led  to  the  discovery  of  the  stimulating  influence  of  all  kinds  of 
fluids  injected  into  the  peritoneal  cavity  of  guinea-pigs. 

In  this  transient  immunity  obtained  with  products  foreign  to  the 
micro-organism  against  which  one  is  vaccinating,  the  most  constant 
and  consequently  most  important  part  is  again  played  by  the  phago- 
cytes. But  there  is  associated  with  it  an  influence,  greater  or  less  in 
degree,  of  substances  present  in  the  serums,  such  as  the  microcytases 
and  fixatives,  which  are  able  to  exercise  a  direct  action  on  the  patho- 
genic micro-organisms.  In  all  cases  known  and  analysed  up  to  the 
present,  the  intervention  of  the  living  organism  of  the  animal  is 
indispensable,  consequently  this  form  of  acquired  immunity  against 
micro-organisms  cannot  be  regarded  as  being  really  passive. 

1  Ztschr.f.  Hyg.,  Leipzig,  1891,  Bd.  xvm,  S.  177. 

2  CentralU.f.  Bakteriol  u.  Parasitenk.,  Jena,  1893,  Bd.  xm,  S.  426. 


CHAPTER  XI  [341] 

NATURAL  IMMUNITY  AGAINST  TOXINS 

Examples  of  natural  immunity  against  toxins. — Immunity  of  spiders  and  scorpions 
against  tetanus  toxin. — Immunity  of  the  scorpion  against  its  own  poison. — 
Antivenomous  property  of  the  blood  of  the  scorpion, — Immunity  against  tetanus 
toxin  in  the  larvae  of  Oryctes  and  in  the  cricket. — Immunity  and  susceptibility 
of  frogs  against  this  toxin. — Natural  immunity  of  reptiles  against  tetanus 
toxin. — Antitetanic  property  of  the  blood  of  alligators. — Immunity  of  snakes 
against  snake  venom. — Immunity  of  the  fowl  against  tetanus  toxin. — Immunity 
of  the  hedgehog  against  poisons  and  venoms. — Immunity  of  the  rat  against 
diphtheria  toxin. 

As  in  this  book  we  are  dealing  specially  with  the  immunity  against 
infective  diseases,  the  question  of  the  resistance  of  the  animal  to 
poisons  interests  us  only  in  so  far  as  it  is  related  to  immunity  against 
micro-organisms.  Consequently  the  reader  must  not  expect  a  treatise 
on  intoxications  properly  so  called  nor  one  on  immunity  against  all 
kinds  of  poisons.  To  perform  such  a  task  we  should  have  to  far 
overstep  the  bounds  of  the  subject  that  we  have  chosen  and  enter 
upon  an  examination  of  questions  which  are  beyond  our  sphere.  Our 
chief  aim  is  to  present  to  the  reader  a  summary  of  our  present 
knowledge  on  immunity  against  microbial  toxins  and  to  establish 
the  relations  between  this  kind  of  immunity  and  immunity  against 
infective  micro-organisms.  In  order  to  do  this,  however,  we  shall 
have  now  and  again  to  go  beyond  the  limits  of  our  programme  and 
discuss  certain  problems  bearing  on  the  resistance  of  the  animal 
organism  against  poisons  not  of  microbial  origin. 

The  immunity  against  toxins,  like  that  against  the  micro-organisms 
themselves,  may  be  either  natural  or  acquired.  As  many  poisons 
have  been  known  from  time  immemorial,  we  are  able  to  collect 
numerous  observations  on  the  resistance  of  the  animal  organism  to 
such  substances  made  when  there  was  no  idea  of  immunity  against 
infective  diseases.  The  etiology  of  intoxications  is  often  much  more 
evident  and  simple  than  is  that  of  infections ;  this  is  one  of  the  [342] 


326  Chapter  XI 

reasons  that  the  older  conceptions  on  the  subject  of  immunity  against 
poisons  were  more  advanced  than  were  those  on  immunity  against 
infective  diseases. 

Several  examples  of  natural  immunity  in  the  lower  animals  have 
already  been  cited.  Thus,  we  have  seen  in  previous  chapters  that  the 
Infusoria  are  resistant  to  poisons  that  exert  a  powerful  action  on  a 
large  number  of  the  higher  animals,  such  as  the  tetanus  and  diph- 
theria toxins  and  especially  the  ichthyotoxin  of  eel's  serum.  We 
have  mentioned  the  case  of  the  larva  of  Oryctes  nasicornis  which  is 
unaffected  by  large  doses  of  the  toxins  of  certain  bacteria  and  which 
at  the  same  time  is  very  subject  to  fatal  infections  by  very  small 
doses  of  the  bacteria  that  form  the  poisons.  These  larvae,  like 
those  of  the  cockchafer,  are,  however,  fairly  susceptible  to  the  poison 
of  the  scorpion.  Several  other  species  of  Arthropoda,  which 
have  been  studied  from  the  point  of  view  of  immunity  against 
toxins,  have  exhibited  analogous  features.  Thus  spiders  and  scor- 
pions are  refractory  to  tetanus  toxin.  In  one  experiment  I  injected 
into  the  abdominal  cavity  of  a  Mygale  from  the  Congo  (which 
weighed  7  grin.  5)  1  c.c.  of  tetanus  toxin  on  two  several  occasions. 
This  dose  is  sufficient  to  kill,  with  the  symptoms  of  tetanus,  1000  mice 
of  double  the  weight.  The  spider,  kept  in  the  incubator  at  36°  C., 
remained  quite  well  during  the  two  months  that  the  experiment 
lasted.  It  exhibited  no  symptom,  not  even  transient,  of  muscular 
stiffening,  nor  any  change  in  its  habits  and  natural  functions.  The 
tetanus  toxin  disappeared  from  the  blood  of  the  Mygale,  but  this 
blood  at  no  time  showed  the  slightest  antitoxic  power  against  this 
poison.  This  example  of  natural  immunity  cannot,  therefore,  be 
ascribed  to  any  antitoxic  property  of  the  fluids  and  must  be  regarded 
as  a  case  of  immunity  of  the  tissues — von  Behring's  histogenic  im- 
munity. In  the  present  imperfect  state  of  our  knowledge  it  is 
impossible  to  describe  precisely  the  mechanism  of  this  immunity. 
When  we  say  that  the  spider  is  refractory  to  the  tetanus  toxin 
because  its  susceptible  elements  have  no  receptors  capable  of  seizing 
the  haptophore  group  of  this  poison,  we  simply  give  expression  to  a 
hypothesis  which  we  are  not  in  a  position  to  verify  by, experiment. 

The  scorpion,  a  well-known  representative  of  the  Arachnida  with 

[343]  segmented  abdomen,  shares  with  the  Mygale  in  the  immunity  against 

tetanus  toxin.    The  Algerian  and  Tunisian  scorpions  (Scorpio  afer  and 

Androctonus  occitanus)  withstand  the  action  of  doses  of  this  poison 

which  are  fatal  for   1000  mice  and  more.     Taking  weight  as  our 


Natural  immunity  against  toxins  327 

standard  we  may  inject  into  them,  with  impunity,  more  than  5000 
times  as  much  toxin  as  into  mice,  without  setting  up  a  single  morbid 
symptom.  Scorpions,  like  the  Mygale,  live  well  in  the  incubator  at 
36°  C.,  where  they  are  kept  whilst  submitted  to  the  action  of  the 
tetanus  poison.  Here  again  we  have  to  do  with  a  case  of  histogenic 
immunity.  The  fluids  of  the  scorpion  exert  no  antitoxic  action. 
When  blood  from  the  normal  scorpion  is  mixed  with  various  doses  of 
tetanus  toxin  and  injected  into  mice  these  animals  contract  tetanus 
and  die  just  as  do  the  control  animals.  In  certain  exceptional  cases 
some  slight  retardation  was  observed,  but  the  blood  of  the  scorpion 
is,  in  most  cases,  incapable  of  preventing  tetanus  in  animals  sus- 
ceptible to  this  disease. 

Scorpions,  injected  with  tetanus  toxin,  do  not  retain  it  in  their 
blood  for  long.  A  few  days  after  the  injection  of  the  tetanus  poison 
such  blood,  when  injected  subcutaneously  into  mice,  excites  no  trace 
of  tetanus.  The  preparation  of  extracts  of  the  different  organs  of 
scorpions  treated  with  tetanus  toxin  demonstrates  that  the  liver  and 
the  liver  only  absorbs  the  poison.  It  is  found  there  a  few  days  after 
the  injection  of  the  toxin  and  it  remains  there  unaltered  for  some 
considerable  time.  The  exudation  of  the  liver  of  scorpions,  killed  a 
month  or  more  after  the  introduction  of  the  toxin  into  the  general 
cavity,  injected  into  mice  sets  up  a  typical  and  fatal  tetanus. 

The  presence  of  the  tetanus  toxin  in  the  organism  of  scorpions 
does  not  give  rise  to  the  production  of  antitoxin.  At  any  rate  a 
whole  series  of  experiments  on  this  point  carried  out  by  us  never 
gave  a  positive  result.  The  scorpions  resisted  repeated  doses  of  the 
tetanus  toxin  and  lived  without  any  difficulty  at  36°  C.,  but  their 
blood  was  never  at  any  period  capable  of  preventing  mice  from 
contracting  fatal  tetanus.  Nevertheless  the  scorpion  may  possess 
antitoxic  power. 

Everyone  has  heard  of  the  supposed  suicide  of  the  scorpion.  We 
are  told  that  when  this  animal  finds  itself  under  conditions  in  which  its 
death  is  inevitable,  it  stings  itself  with  the  end  of  its  tail  and  dies 
from  the  effect  of  its  own  poison.  A  simple  method  of  reproducing  [344] 
this  experiment  is  actually  described : — Surround  the  scorpion  with  a 
circle  of  fire.  The  animal  rushes  in  all  directions  to  find  a  way  out, 
and  finding  none,  deliberately  commits  suicide.  Bourne1  at  Madras 
carefully  investigated  this  question  in  a  large  species  of  Indian 

1  Proc.  Roy.  Soc.  London,  1887,  Vol.  XLII,  p.  17. 


328  Chapter  XI 

scorpion  and  demonstrated  the  absolute  erroneousness  of  the  story 
of  suicide  which,  had  it  been  true,  would  have  afforded  a  unique 
example  of  voluntary  death  in  animals.  On  carrying  out  the  classic 
experiment  he  observed  that  within  this  ring  of  fire  the  scorpion 
is  subjected  to  a  very  high  temperature.  When  the  temperature 
reaches  40°  C.  the  scorpion  begins  to  grow  weak  and  as  the  tempera- 
ture approaches  50°  C.  it  passes  into  a  comatose  condition.  Moreover 
Bourne  showed  that  the  scorpion's  poison,  which  is  fatal  for  large 
spiders,  insects,  and  vertebrates,  was  innocuous  for  individuals  of  the 
species  furnishing  it. 

I  can  confirm  all  the  statements  of  this  English  observer. 
When  I  was  studying  the  embryology  of  the  scorpion  I  repeatedly 
tried  the  experiment  but  the  animal  never  committed  suicide. 
Further,  I  repeatedly  assured  myself  of  the  innocuousness  of  the 
scorpion's  poison  when  injected  into  individuals  of  the  same  species, 
and  I  was  able  to  demonstrate  most  conclusively  that  the  blood  of  the 
scorpion  is  endowed  with  undoubted  antitoxic  power.  The  addition 
of  0*1  c.c.  of  this  blood  to  a  dose  of  poison  which  kills  mice  in  half- 
an-hour  is  sufficient  to  enable  a  mouse  injected  with  the  mixture  to 
resist  it  completely.  This  antitoxic  power  is  the  same  in  the  Scorpio 
afer  and  in  the  Algerian  Androctonus.  An  emulsion  of  the  liver  of 
the  scorpion,  however,  is  absolutely  incapable  of  preventing  fatal 
intoxication  of  mice. 

This  case  of  antitoxic  action  is  the  only  one  I  have  been  able  to 
demonstrate  in  an  invertebrate.  Must  we  regard  it  as  a  case  of 
natural  innate  antivenomous  power  or  as  something  acquired  during 
the  life  of  the  animal?  It  is  not  easy  to  settle  this  question  by 
experiment  We  can  certainly  procure  new-born  scorpions  and  rear 
them  for  some  time,  but  the  quantity  of  blood  that  can  be  got  from 
them  is  insufficient  for  injection  for  protective  purposes.  Scorpions 
do  not  love  one  another  and  when  kept  together  we  often  find  them 
engaged  in  fierce  and  mortal  combat,  the  stronger  killing  the  weaker 
and  sucking  their  blood.  It  is  therefore  possible  that,  in  some  stage 
of  their  life,  the  scorpions  find  means  of  vaccinating  themselves 
against  their  own  poison  either  through  the  intestine  or  as  the  result 
[345]  of  punctures  caused  by  the  point  of  the  tail.  It  would  be  very 
interesting  to  study  this  question  under  favourable  conditions,  because 
it  is  capable  of  throwing  light  on  the  problem  of  the  origin  of  anti- 
toxins, from  a  general  point  of  view.  Whichever  view  be  taken,  the 
acquisition  of  any  antitoxic  property  by  the  blood  in  the  Invertebrata 


Natural  immunity  against  toxins  329 

must  take  place  slowly  and  with  great  difficulty  as  is  shown  by  our 
want  of  success  with  tetanus  toxin. 

Insects  are,  as  a  rule,  very  tolerant  of  this  latter  poison.  As, 
however,  the  tetanus  toxin  (we  shall  illustrate  this  later)  only  acts 
well  and  in  small  doses  at  a  high  temperature  (about  30°  C.)  and  as 
most  insects  do  not  readily  adapt  themselves  to  this  temperature, 
it  was  necessary  to  choose  species  capable  of  living  at  these  high 
temperatures  and  for  this  line  of  study  the  larva  of  Oryctes  is  most 
suited.  It  flourishes  well  at  a  temperature  of  30° — 36°  C.,  and  under 
these  conditions  exhibits  a  much  greater  resistance  to  infection  by 
Isaria  than  at  lower  temperatures.  It  can  be  kept  in  the  incubator 
for  months  if  placed  in  glass  jars  filled  with  earth  mixed  with  tanner's 
bark.  The  injection  of  enormous  quantities  of  very  active  tetanus 
toxin  directly  into  the  blood  has  not  the  slightest  effect  on  these 
larvae.  Whilst,  however,  the  blood  fluid  of  the  Arachnida  rapidly 
gets  rid  of  the  poison,  that  of  Oryctes  retains  it  for  a  very  long  period. 
If  a  small  quantity  of  blood  be  taken  from  larvae  several  months 
after  injection  and  then  injected  into  mice,  these  animals  contract 
typical  tetanus  and  quickly  succumb. 

The  toxin,  however,  finally  disappears  from  the  blood  though  a 
certain  portion  of  it  may  still  be  found  in  the  pericardial  cells  and 
especially  in  the  fat-bodies. 

Never,  under  any  circumstances,  was  I  able  to  observe  that  the 
blood  of  the  larvae  of  Oryctes  exerted  any  antitoxic  action.  At  the 
stage  when  this  fluid  no  longer  gives  tetanus  to  mice,  it  is  absolutely 
incapable  of  preventing  intoxication  when  mixed,  before  injection, 
with  tetanus  toxin. 

Amongst  adult  insects  the  cricket  is  best  adapted  for  researches 
on  tetanus.  The  field  cricket  will  bear  a  temperature  even  higher 
than  30°  C.  It  is  completely  resistant  to  injections  of  tetanus  toxin, 
but  it  showed  no  more  capacity  than  did  the  larvae  of  Oryctes  or  the 
Arachnida  of  producing  any  tetanus  antitoxin. 

All  the  Invertebrata  that  I  have  been  able  to  study  have  exhibited 
a  remarkable  resistance  against  the  known  bacterial  toxins,  but  the  [346] 
mechanism  of  this  natural  immunity  could  not  be  exactly  made  out 
owing  to  the  difficulty  met  with  in  investigating  the  toxins  in  the 
organs  and  following  their  modifications.  The  idea  of  making  use  of 
these  lower  animals  for  the  purpose  of  solving  the  problem  of  the 
origin  of  antitoxins  is  not  realisable,  from  the  fact  that  the  Inverte- 
brata that  have  been  studied  have  never,  in  my  experience,  produced 


330  Chapter  XI 

any  of  these  substances  as  the  result  of  injections,  whether  single  or 
repeated,  of  toxins. 

The  natural  immunity  of  the  Invertebrata  against  bacterial  toxins 
cannot  therefore  be  regarded  as  an  example  of  humoral  immunity. 
It  must  be  placed  in  the  category  of  histogenic  immunity,  although 
we  are  not  in  a  position  to  define  accurately  the  part  played  by  the 
cellular  elements  in  the  defence  of  the  animal  against  these  poisons. 
We  must,  therefore,  go  higher  up  in  the  animal  scale  if  we  are  to 
solve  the  principal  questions  in  regard  to  antitoxic  immunity. 

The  lowest  Vertebrata,  the  fishes,  are  not  well-suited  for  this  kind 
of  research.  The  best  known  bacterial  toxins  act  specially  on  warm- 
blooded animals  and  require  the  co-operation  of  high  temperatures. 
Fishes  do  not  live  well  in  captivity  except  at  relatively  low  tempera- 
tures and  soon  die  if  placed  in  an  incubator  kept  at  30°  C.  or 
higher.  It  is  necessary,  therefore,  to  have  recourse  to  the  Amphibia, 
which  are  much  more  easily  acclimatised  to  these  temperatures.  The 
Axolotl,  coming  from  Mexico,  is  naturally  capable  of  withstanding  great 
heat.  These  animals  will  live  for  long  at  a  temperature  of  30° — 37°  C. 
They  possess  the  drawback,  however,  of  being  very  susceptible  to  the 
tetanus  toxin,  very  small  doses  of  it  being  fatal.  The  green  frog 
(Rana  esculentci)  is  the  most  suitable  for  our  purpose.  It  readily 
adapts  itself  to  optimum  temperatures  (30° — 36°  C.)  and  exhibits  at 
least  a  certain  degree  of  immunity  against  various  bacterial  toxins. 
We  have  stated  in  a  preceding  chapter  that  the  green  frog  is  un- 
affected by  considerable  quantities  of  diphtheria  toxin.  It  is 
resistant  also  to  tetanus  toxin,  but  this  natural  immunity  appears  to 
be  connected  with  special  conditions.  Courmont  and  Doyon1  were 
[347]  the  first  to  draw  attention  to  the  fact  that  beyond  20° — 25°  C.  green 
frogs  may  contract  tetanus.  Refractory  in  winter  they  become  sus- 
ceptible in  summer.  These  observers  afterwards  found  that  of  frogs 
inoculated  with  the  same  dose  of  toxin  and  divided  into  two  sets,  one 
set  kept  at  a  temperature  of  about  10°  C.  remained  quite  well  whilst 
the  other  set  subjected  to  one  of  30° — 39°  C.  contracted  tetanus  after 
five  days'  incubation.  This  experiment  has  been  confirmed  by  several 
observers,  and  indicates  that  the  tetanus  poison  demands,  for  the 
manifestation  of  its  toxic  action,  a  favourable  and  fairly  high  tempera- 
ture. This  result  must,  however,  be  accepted  with  some  reserve. 
Undoubtedly  the  doses  of  tetanus  toxin  which  induce  fatal  tetanus  in 

1  Compt.  rend.  Soc.  de  biol.y  Paris,  1893,  pp.  294,  618;  1898, p.  344.  "Le  tetanos," 
Paris,  1899,  p.  25. 


Natural  immunity  against  toxins  331 

frogs  kept  at  a  high  temperature  are  innocuous  when  these  animals 
are  living  at  low  temperatures.  But  we  can,  by  increasing  the  dose, 
produce  tetanus  in  frogs  even  when  the  temperature  is  not  very  high. 
Thus  Marie1  was  able,  during  the  whole  of  the  winter,  to  tetanise 
both  green  and  brown  frogs  living  in  water  the  temperature  of  which 
oscillated  between  13°  and  18°  C.  The  incubation  period  in  this  case 
is  very  much  longer  (sometimes  extending  to  25  days)  than  in  frogs 
kept  at  higher  temperatures. 

Temperature,  therefore,  is  an  important  factor  in  the  poisoning  by 
the  tetanus  toxin  and  in  the  resistance  of  the  frog,  but,  in  the  long 
run,  this  poison  can  exert  its  specific  action  even  at  relatively  low 
temperatures. 

Morgenroth2  endeavoured  to  analyse  the  mechanism  of  this  re- 
sistance and  of  the  susceptibility  of  the  green  frog  when  maintained 
at  various  temperatures.  He  demonstrated  that  the  tetanus  toxin  is 
fixed  in  the  central  nervous  system,  even  at  low  temperatures,  near 
8°  C. ;  under  these  conditions,  however,  it  is  incapable  of  causing  the 
slightest  tetanic  symptom.  When  placed  in  an  incubator  kept  at 
32°  C.  the  frogs  contract  tetanus  after  a  period  of  incubation  of  some 
(2  to  3)  days.  During  the  first  24  hours  of  this  period  the  frogs 
manifest  no  sign  of  tetanus,  and  if  they  are  again  put  in  a  cool 
place  they  continue  in  good  health.  If,  however,  after  a  not  too  pro- 
longed stay  in  the  cold,  these  animals  are  subjected  a  second  time  to  [348] 
the  higher  temperature,  they  become  tetanic,  after  a  shortened 
incubation  period.  Cold,  therefore,  may  arrest  tetanus  even  at  a  stage 
when  the  toxin  has  already  produced  certain  latent  but  permanent 
modifications  of  the  nervous  system. 

Frogs  injected  with  tetanus  toxin  and  kept  in  a  cold  place  finally 
get  rid  of  the  poison.  When  transferred  to  a  warm  chamber  after 
a  certain  lapse  of  time  they  no  longer  contract  tetanus.  We  have 
found  that  the  greater  part  of  the  tetanus  toxin  continues  for  some 
time  in  the  blood  of  frogs  injected  and  kept  at  a  low  temperature.  A 
small  quantity  of  this  blood  withdrawn  almost  two  months  after  the 
last  injection  produced  fatal  tetanus  in  a  mouse.  We  do  not  know 
how  frogs  eliminate  the  toxin,  but  it  has  been  demonstrated  that  in 
this  case  it  causes  no  production  of  antitoxin.  Morgenroth  has  con- 
firmed this  result. 

Reptiles  must  be  regarded   as  vertebrates    exhibiting   a  most 

1  Ann.  de  Vlnst.  Pasteur,  Paris,  1897,  t  xi,  p.  597. 

2  Arch,  internat.  de  Pliarmacodyn.,  Gand  et  Paris,  1900,  VoL  vn,  p.  265. 


332  Chapter  XI 

pronounced  natural  immunity  against  tetanus.  They  show  an  un- 
limited resistance  to  enormous  doses  of  tetanus  poison,  and  this  at 
low,  medium,  or  high  temperatures  (30° — 37°  C.).  Green  lizards  with- 
stand considerable  doses  of  tetanus  toxin.  Although  they  do  not 
contract  tetanus,  they  get  rid  of  the  poison  exceedingly  slowly.  Thus, 
a  lizard  kept  at  a  temperature  of  20°  C.,  and  injected  with  an  amount 
of  toxin  sufficient  to  kill  500  mice,  at  the  end  of  two  months  still 
retains  in  its  blood  such  an  amount  of  the  poison  that  one-tenth  of  a 
c.c.  will  cause  fatal  tetanus  in  a  mouse.  Turtles  present  an  analogous 
case.  The  marsh  turtle,  Emys  orbiculariSj  tolerates  very  large 
amounts  of  tetanus  toxin,  injected  subcutaneously,  and  this  at  both 
low  and  high  temperatures,  at  30°  C.  and  beyond  (36°— 37°  C.).  The 
toxin  passes  quickly  into  the  blood  and  remains  localised  there  for  a 
very  long  time.  In  a  turtle  kept  in  an  aquarium  at  the  laboratory 
the  blood  was  tetanigenic  for  the  mouse  even  four  months  after  an 
intra-peritoneal  injection  of  the  toxin.  In  another  turtle  which  lived 
at  incubator  temperature  (36° — 37°  C.),  the  blood  was  still  toxic  two 
months  after  a  subcutaneous  injection  of  tetanus  toxin  in  quantity 
fatal  for  500  mice.  In  turtles  kept  at  36°  C.  I  observed  abundant 
transudations  into  the  peritoneal  cavity,  and  the  fluid,  very  poor  in 
[349]  formed  elements,  was  found  to  be  very  tetanigenic.  It  must  be 
accepted,  therefore,  that  the  toxin  is  retained  in  the  blood  plasma 
with  which  it  passes  into  the  trausudation.  Every  kind  of  cell  must 
exhibit  a  very  marked  negative  chemiotaxis  against  tetanus  toxin  for 
this  poison  to  be  retained  so  long  in  the  body  fluids.  Under  these 
conditions  it  is  not  surprising  that  in  turtles  I  was  never  able  to 
observe  the  slightest  antitoxic  power  in  the  blood.  Their  great 
natural  immunity  must  be  due  to  some  other  factor. 

The  alligator  (Alligator  mississippiensis)  has  also  been  found  to 
be  quite  refractory  to  tetanus  both  at  low  and  at  high  temperatures. 
Outwardly  alligators  behave  exactly  as  do  turtles,  that  is  to  say,  after 
the  injection  of  various  and  sometimes  very  large  doses  of  toxin  they 
exhibit  no  morbid  symptom  either  general  or  tetanic.  But  the  par- 
ticular changes  which  occur  in  their  organism  differ  essentially  from 
those  met  with  in  the  turtle.  The  toxin  is  rapidly,  eliminated  from 
the  blood  of  the  alligator,  even  when  it  is  kept  at  a  relatively  low 
temperature  (20°  C.).  Under  these  conditions  of  temperature,  how- 
ever, the  blood  does  not  become  antitoxic  although  it  has  lost  its 
tetanigenic  property.  When,  however,  the  alligators  are  kept  at  a 
higher  temperature  (32°— 37°  C.),  antitoxic  power  is  developed  in 


Natural  immunity  against  toxins  333 

their  blood,  often  with  very  great  rapidity.  Quite  young  alligators 
(weighing  about  500  grammes)  are  capable  of  producing  antitoxin, 
though  somewhat  slowly.  A  month  after  the  first  injection  of  the 
tetanus  toxin  their  blood  is  incapable  of  causing  tetanus  in  mice,  but 
is  not  yet  antitoxic.  A  month  later,  however,  it  never  fails  to  prevent 
an  attack  of  tetanus  when  mixed  with  fatal  doses  of  the  toxin  and 
injected  into  mice. 

Older  alligators  develop  antitoxic  power  much  more  rapidly,  and 
on  several  occasions  we  have  found,  to  our  great  astonishment,  that, 
as  early  as  24  hours  after  injection  of  the  toxin,  their  blood  was 
distinctly  antitetanic.  The  blood  of  the  same  alligators,  tested  before 
the  injection  of  the  toxin,  like  the  blood  of  normal  alligators 
generally,  exhibited  no  antitoxic  property. 

In  several  experiments  we  took  the  rectal  temperature  of  our 
animals  and  were  never  able  to  observe  the  slightest  rise  correspond- 
ing to  the  temperature  of  the  water  in  which  the  alligators  lived. 

It  cannot  be  doubted  then,  that,  in  spite  of  the  facility  with  which  [350] 
these  reptiles  produce  tetanus  antitoxin,  their  immunity  does  not 
depend  on  this  antitoxic  property.  Thus,  young  alligators  which 
have  resisted  a  single  dose  of  toxin  sufficient  to  kill  6000  mice  must 
owe  their  immunity  to  some  other  cause  than  the  antitoxic  power  of 
the  body  fluids,  for  their  blood  does  not  begin  to  exhibit  this 
property  until  two  months  after  injection. 

These  same  reptiles  are  also  very  refractory  against  cholera  toxin, 
even  in  large  doses  ;  they  react  to  the  injection  by  the  development 
of  the  corresponding  antitoxin.  On  the  other  hand  they  are  very 
susceptible  to  diphtheria  toxin,  small  quantities  of  which  are  quite 
sufficient  to  bring  about  a  fatal  intoxication. 

Snakes,  like  other  reptiles,  are  refractory  against  tetanus  toxin. 
In  the  study  of  their  natural  immunity,  however,  we  are  confronted 
by  the  difficulty  that  their  blood  is  naturally  toxic  for  laboratory 
animals.  This  toxin,  analogous  to  the  ichthyotoxin  of  eel's  serum,  has 
been  compared  with  snake  venom  against  which  the  snakes  them- 
selves enjoy  a  very  marked  immunity. 

Xot  venomous  snakes  only  exhibit  immunity  against  their  own 
poison.  Long  ago  Font  ana1  observed  that  non-veDomous  snakes 
resist  the  bite  of  the  viper  and  even  subcutaneous  inoculation  of 
its  venom.  Phisalix  and  Bertrand2  confirmed  these  observations 

1  "  Traite  sur  le  venin  de  la  vipSre,"  Florence,  1781. 

3  Arch,  dephysiol  norm,  et  path.,  Paris,  Annee  xxvi,  1894,  p.  423. 


334  Chapter  XI 

and  were  able  to  show  that  a  non- venomous  snake  (Tropidono- 
tus)  will  withstand  a  dose  of  venom  capable  of  killing  from  15  to 
20  guinea-pigs.  Seeking  for  the  cause  of  this  natural  immunity, 
these  observers  came  to  the  conclusion  that  it  is  due  to  the 
presence  in  the  blood  of  toxic  substances  analogous  to  those  of  the 
venom  of  the  viper.  These  same  substances  are  found  also  in  the 
labial  glands  of  the  upper  jaw  of  the  Tropidonotus  and  can  from 
thence,  according  to  the  view  of  Phisalix  and  Bertrand,  pass  into  the 
blood  as  an  internal  secretion.  Calmette1  has  shown  that  the  blood 
of  snakes,  injected  in  a  non-toxic  dose,  vaccinates  certain  mammals 
against  snake  venom,  and  Phisalix  and  Bertrand  have  even  obtained 
an  antitoxic  effect  by  injecting  a  mixture  of  snake's  blood,  heated  to 
58°  C.,  with  lethal  doses  of  venom.  There  is,  then,  in  this  example 
[351]  something  analogous  to  what  we  have  described  in  scorpions,  with 
this  difference,  however,  that  the  blood  of  these  Arachnids  is  already 
antitoxic,  to  a  certain  degree,  whilst  that  of  snakes  only  becomes 
so  after  it  has  been  modified  by  heat. 

The  classic  example  of  immunity  against  a  bacterial  toxin 
amongst  Birds  is  that  of  the  fowl,  which  is  highly  refractory  against 
the  tetanus  toxin.  In  the  very  earliest  researches  on  this  poison 
injections  were  made  into  vertebrates  of  very  different  kinds,  and  a 
very  striking  feature  was  the  facility  with  which  fowls  resist  very 
large  quantities  of  tetanus  toxin.  However,  as  is  almost  always  the 
case,  this  immunity  has  been  found  not  to  be  absolute.  By  means  of 
enormous  doses,  injected  subcutaneously  or  into  the  muscular  tissue, 
tetanus  of  the  most  typical  kind,  ending  in  death,  has  been  induced  in 
fowls,  and  in  fowls  weakened  by  cold,  tetanic  intoxication,  even  with 
smaller  doses,  has  been  set  up.  By  injecting  the  toxin  directly  into 
the  brain,  according  to  Roux  and  Borrel's  method,  the  fowl  may  be 
still  more  easily  tetanised.  Thus,  von  Behring2  observed  that  by 
injecting  one  milligramme  of  the  toxin  into  the  brain  of  a  fowl, 
weighing  one  kilo,  tetanus  may  infallibly  be  produced. 

After  the  brilliant  and  fruitful  discovery  of  the  antitoxic  property 
of  the  blood,  made  by  von  Behring  in  collaboration  with  Kitasato, 
we  were  justified  in  concluding  that  immunity  against  toxins  and, 
amongst  others,  natural  immunity,  might  depend  on  the  power  of  the 
body  fluids  to  neutralise  the  toxins.  This  hypothesis  has  been  formu- 
lated at  various  times,  but  it  was  for  the  first  time  subjected  to 

"  Le  venin  des  serpents,"  Paris,  1896,  p.  40. 

"  Allgemeine  Therapie  der  Infectionskrankheiten,"  Berlin  u.  Wien,  1899,  S.  992. 


Natural  immunity  against  toxins  335 

experimental  control  by  Vaillard1,  and  specially  in  connection  with 
tetanus  in  the  fowl.  The  blood  or  blood  serum  of  these  birds,  when 
mixed  in  varying  doses,  small,  medium,  and  large,  with  tetanus  toxin, 
was  never  found  to  be  capable  of  preventing  susceptible  animals 
(mice,  guinea-pigs,  rabbits)  from  contracting  tetanus :  these  animals 
so  treated  behaved  just  as  did  the  controls  inoculated  with  toxin 
only. 

The  great  resistance  of  the  fowl  against  tetanus, — one  of  the  most 
typical  examples  of  natural  immunity  against  a  microbial  poison,— 
cannot,  therefore,  be  explained  by  the  presence  in  the  body  fluids  of 
an  antitoxin  capable  of  neutralising  and  rendering  innocuous  the  [352] 
tetanus  toxin.  On  the  other  hand,  we  are  not  justified  in  attributing 
it  simply  to  the  absence  of  corresponding  receptors  in  the  sensitive 
nerve  cells.  Since  the  fowl  readily  contracts  tetanus  when  the  toxin 
is  injected  directly  into  the  brain  or  when  the  fowl  is  weakened  by 
cold,  it  is  evident  that  the  sensitive  elements  never  fail  to  absorb  and 
fix  any  poison  that  is  presented  to  them.  In  ordinary  cases,  however, 
when  the  fowl  exhibits  its  remarkable  resisting  power  against  the 
toxin  injected  in  very  large  quantity,  subcutaneously,  into  the  muscles 
or  into  the  peritoneal  cavity,  the  poison  does  not  reach  the  sensitive 
cells,  being  arrested  and  rendered  innocuous  whilst  circulating  in  the 
tissues  of  the  organism. 

Yon  Behring2  is  of  opinion  that  in  examples  of  natural  immunity, 
such  as  the  one  just  examined,  the  principal  cause  of  the  refrac- 
tory condition  depends  upon  the  impermeability  to  the  toxin  of  the 
capillary  wall  of  the  vessels.  It  is,  however,  difficult  to  maintain  this 
thesis  in  regard  to  tetanus  in  the  fowl,  when  it  is  remembered  how 
readily  tetanus  toxin  passes  through  filters  and  membranes,  and 
especially  in  view  of  the  fact  that  weakening  of  the  fowl  by  means  of 
cold  renders  it  susceptible  to  doses  of  toxin  which  are  tolerated 
without  inconvenience  by  normal  fowls. 

We  are,  therefore,  compelled  to  place  the  natural  immunity  of  the 
fowl  against  tetanus  toxin  in  the  category  of  cell  immunities.  This 
toxin,  as  we  have  said,  must  be  arrested  en  route  before  it  reaches  the 
cells  of  the  nerve  centres.  But  where  and  how  does  this  beneficent 
arrest  take  place?  Ten  years  ago  Vaillard  demonstrated  that  the 

1  Compt.  rend.  Soc.  de  Mol,  Paris,  1891,  p.  462;  Ann.  de  VInst.  Pasteur,  Paris, 
1892,  t  VI,  p.  229. 

2  Article :  Infectionsschutz  und  Immunitat  in  Eulenburg's  "  Real-encyclopadie 
d.  ges.  Heilkunde"  (Encyclop.  Jahrbiicher),  Wien,  1900,  Bd.  IX,  S.  203. 


336  Chapter  XI 

blood  of  fowls  that  have  received  an  injection  of  tetanus  toxin  causes 
typical  tetanus  in  susceptible  animals.  This  tetanigenic  property  of 
the  blood  persists  for  a  certain  number  of  days.  When  it  is  measured 
by  the  quantitative  method,  it  is  found  that  all  or  almost  all  the 
tetanus  toxin  injected  into  the  peritoneal  cavity  of  the  fowl  passes 
into  the  blood  and  remains  there  intact  for  a  variable  number  of  days. 
From  a  morphological  point  of  view  the  blood,  immediately  after  the 
injection  of  the  toxin,  exhibits  a  hyperleucocytosis  of  greater  or  less 
duration. 

When  the  fowls  are  killed  at  the  stage  when  their  blood  becomes 
tetanigenic  (as  the  result  of  the  injection  of  the  toxin  into  the  peri- 
[353]  toneal  cavity),  it  can  be  demonstrated  that  their  viscera  are  not 
capable  of  producing  tetanus  in  susceptible  animals  except  in  so  far 
as  they  contain  blood.  It  is  only  the  vascular  organs,  rich  in  bloodt 
such  as  the  spleen,  liver,  kidneys,  thyroid  gland  and  bone-marrow, 
that  impart  tetanus  and  then  only  in  so  far  as  they  have  not  been 
freed  from  blood.  Of  the  various  organs  only  the  genital  glands, 
ovaries  or  testes,  absorb  a  certain  amount  of  the  injected  toxin.  Very 
young  testes  or  the  smallest  ovarian  ova  containing  as  yet  no  trace  of 
yellow  yolk,  when  injected  into  mice,  produce  a  fatal  tetanus. 

In  fowls,  insusceptible  to  tetanus  toxin,  this  toxin  is  found, 
then,  in  the  sexual  glands  and  in  the  blood.  When,  in  order 
to  ascertain  the  -exact  localisation  of  this  toxin,  we  measure  the 
tetanigenic  power  of  the  whole  blood  as  compared  with  that  of  the 
aseptic  exudations  induced  by  the  injection  of  gluten-casein,  and 
necessarily  much  richer  in  leucocytes,  we  get  the  result  that  the  ex- 
udations contain  more  tetanus  toxin  than  does  the  blood.  We  are 
led,  therefore,  to  the  conclusion  that  this  poison  is  absorbed,  at  least 
in  part,  by  the  leucocytes,  and  it  is  in  these  elements  and  in  the 
genital  cells  that  we  must  look  for  the  factors  which  arrest  the  toxin 
and  prevent  its  reaching  the  nerve  centres. 

Cellular  or  histogenic  immunity  is  often  contrasted  with  chemical 
immunity  without  taking  into  consideration  the  real  analogies  and 
differences  to  be  found  between  them.  It  is  evident  that  in  both 
groups  the  organism  of  the  animal  modifies  the  introduced  toxins  and 
that  this  modification  is  a  chemical  process.  In  cellular  immunity, 
however,  this  act  is  preceded  by  certain  biological  phenomena,  such 
as  the  reaction  of  the  formed  elements  and  the  absorption  of  the 
noxious  substance.  Immunity  in  these  cases  is  more  complex  than  in 
the  example  where  the  toxin  is  neutralised  by  a  direct  action  of  the 


Natural  immunity  against  toxins  337 

body  fluids,  but  ultimately  it  always  resolves  itself  into  a  chemical  or 
perhaps  physico-chemical  action  of  the  substances  of  the  organism  of 
the  animal  on  the  toxic  substances  of  the  poisons. 

In  Mammals  examples  of  natural  immunity  against  certain 
poisons  are  not  rare.  Almost  a  century  ago  Oken  made  the  obser- 
vation that  a  person  who  tried  to  poison  a  hedgehog  with  opium, 
hydrocyanic  acid,  arsenic  or  mercury  bichloride  usually  failed  in  his 
attempts  because  of  the  great  resisting  power  of  this  animal.  Harnack 
demonstrated  that  the  hedgehog  will  withstand  a  dose  of  potassium 
cyanide  six  times  as  great  as  that  necessary  to  kill  a  cat  in  a  few  [354] 
minutes  (0*01  grm.).  In  LewinV  experiments  the  hedgehog  was 
found  to  resist  the  injection  of  powdered  cantharides  in  a  quantity 
seven  times  as  great  as  that  which  infallibly  kills  a  dog  and  greater 
also  than  the  lethal  dose  for  man.  The  same  observer  also  confirms 
the  observation  that  a  much  larger  dose  of  alcohol  must  be  used  in 
order  to  intoxicate  a  hedgehog  than  is  required  to  obtain  the  same  effect 
in  the  rabbit  or  even  in  the  dog.  Horvath2  fed  hedgehogs  for  a  fairly 
long  period  with  living  cantharides.  These  Insectivora  devour  their 
venomous  prey  without  showing  any  sign  of  illness  except  a  certain 
degree  of  emaciation.  When  Lewin  tried  to  ascertain  the  cause  of 
this  natural  immunity  of  the  hedgehog  he  examined  the  blood  of 
this  animal  for  a  substance  antitoxic  to  cantharidine.  His  experi- 
ments were  all  negative  ;  but  it  is  difficult  to  come  to  any  definite 
conclusion  in  this  matter  from  the  fact  that  the  blood  and  blood 
serum  of  the  normal  hedgehog  are  toxic  for  the  small  laboratory 
animals.  A  similar  objection  had  already  been  brought  forward  by 
Phisalix  and  Bertrand  in  connection  with  their  experiments,  analogous 
to  those  of  Lewin,  on  the  immunity  of  the  hedgehog  against  the 
venom  of  the  viper. 

It  has  long  been  known  that  hedgehogs  have  a  liking  for  certain 
reptiles  and  wage  an  implacable  war  on  snakes  in  general  and  on  the 
viper  in  particular.  In  its  attack  the  hedgehog  tries  to  avoid  being 
bitten,  but  when,  as  often  happens,  it  fails  to  evade  a  bite  the 
inoculation  of  the  viper's  venom  appears  to  be  well  borne.  This 
observation  has  been  confirmed  experimentally.  Phisalix  and 
Bertrand3  have  shown  that  the  resistance  of  the  hedgehog  to  the 


1  Deutsche  med.  Wchnschr.,  Leipzig,  1898,  S.  373. 

2  Vrach,  St  Petersburg,  1897,  p.  964. 

3  Compt.  rend.  Soc.  de  biol.,  Paris,  1899,  p.  77;  Bull.  Museum  d.  hist,  nat.,  Paris, 
1895,  t.  i,  p.  294. 

B.  22 


338  Chapter  XI 

viper's  venom  is  about  forty  times  as  great  as  that  of  the  guinea-pig, 
that  is  to  say  the  hedgehog,  though  far  from  possessing  an  absolute 
immunity,  nevertheless  exhibits  a  much  greater  resistance  than  do 
most  animals.  Lewin1  convinced  himself  of  this  fact  as  regards 
adult  hedgehogs,  though  young  animals,  according  to  him,  are  much 
[355]  more  susceptible.  Thus,  he  has  seen  a  young  hedgehog  that  had 
been  bitten  by  a  viper  die  after  nine  days'  illness.  This  observation 
speaks  in  favour  of  the  conclusion  that  the  immunity  of  the  hedge- 
hog might  be  naturally  acquired  rather  than  a  really  natural  im- 
munity. The  hedgehog,  hunting  all  kinds  of  small  animals,  might 
often  be  bitten  by  vipers  and  in  this  way  acquire  its  immunity 
against  the  venom.  Under  these  conditions  we  can  readily  conceive 
that  the  blood  of  this  "  insectivoran  "  might  be  placed  in  a  position 
to  develop  a  specific  antitoxic  property. 

When  Lewin  tried  to  satisfy  himself  of  the  existence  of  this 
property  by  direct  experiment  he  could  only  show  that  the  blood 
of  the  hedgehog  was  powerless  to  prevent  the  lethal  effect  of  the 
viper's  venom  on  small  animals.  But  here,  as  in  his  researches  on 
cantharidine,  he  did  not  take  into  account  the  inherent  toxicity  of 
the  blood  of  the  hedgehog.  Phisalix  and  Bertrand2,  who  have 
also  studied  this  question,  have  obtained  results  at  variance  with 
those  of  Lewin.  They  demonstrated  first  of  all  that  the  blood  of 
normal  hedgehogs  was  capable  of  intoxicating  and  even  of  killing 
laboratory  animals  such  as  the  guinea-pig.  It  is  quite  natural,  there- 
fore, that  the  mixture  of  this  fluid  with  viper's  venom  could  not  be 
tolerated.  It  was,  however,  sufficient  to  heat  the  blood  of  the  hedge- 
hog to  58°  C.  for  it  to  become  not  only  innocuous  of  itself,  but  even 
for  it  to  exhibit  an  antitoxic  action  against  snake  venom.  Thus, 
guinea-pigs  which  had  received  8  c.c.  of  heated  hedgehog's  serum 
into  the  peritoneal  cavity,  were  at  once  in  a  condition  to  resist  double 
the  lethal  dose  of  viper's  venom.  Phisalix  and  Bertrand  conclude, 
therefore,  that  "  the  natural  immunity  of  the  hedgehog  against  the 
viper's  venom  is  due  to  the  presence  in  its  blood  of  an  immunising 
substance."  The  same  observers3  satisfied  themselves  that  horse's 
serum  and  even  that  of  the  guinea-pig  exercise  an  undoubted  anti- 
.  venomous  action  ;  yet  these  animals  are  anything  but  insusceptible  to 
snake  venom.  Moreover,  the  necessity  to  heat  the  blood  to  58°  C.,  as  a 

1  Deutsche  med.  Wchnschr.,  Leipzig,  1898,  S.  629. 

2  Compt.  rend.  Soc.  de  biol.,  Paris,  1895,  p.  639. 

3  Bull  Museum  d'hist.  nal.,  Paris,  1896,  t.  II,  p.  100. 


Natural  immunity  against  toxins  339 

preliminary  measure,  deprives  this  conclusion  of  the  degree  of  certainty 
one  would  like  to  have  in  such  a  matter.  On  the  other  hand,  the 
greater  susceptibility  of  young  hedgehogs  prevents  us  from  putting 
the  immunity  of  the  t  adult  in  the  category  of  natural  immunity 
properly  so  called. 

Analogous  considerations  apply  in  the  case  of  the  mongoose  [356] 
(Herpestes  ichneumon),  carefully  studied  by  Calmette1,  according 
to  whose  researches  the  Antilles  mongoose  is  not  very  susceptible 
to  snake  venom;  it  readily  withstands  doses  very  large  relatively 
to  its  size,  but  its  immunity  is  not  absolute.  It  owes  much  of  its 
mastery  in  its  fights  with  venomous  snakes  to  its  extraordinary 
agility.  The  blood  of  the  mongoose,  mixed  with  venom,  exhibits  an 
undoubted  antitoxic  power,  though  this  is  not  sufficient  to  prevent 
the  death  of  susceptible  animals.  We  have  no  data  to  enable  us  to 
explain  the  origin  of  this  antitoxic  property,  but  it  is  probable  that 
here  again  we  have  an  example  of  relative  immunity,  acquired  during 
life.  Calmette  points  out,  however,  that  his  ichneumons  came  from 
Guadeloupe,  where  no  venomous  snakes  are  found.  We  may,  of 
course,  suppose  that  the  feebly  antitoxic  power  of  the  blood  of  these 
mammals  might  be  due  to  other  snakes  or  to  species  of  animals 
whose  blood  possesses  a  certain  venomous  property2. 

We  have  far  more  exact  data  on  the  natural  immunity  of  certain 
mammals  against  toxins  of  microbial  origin.  The  example  most 
thoroughly  studied,  one  which  has  become,  one  might  say,  classic,  is 
that  of  the  rat  against  diphtheria  toxin.  Since  the  discovery  of  this 
toxin,  the  first  well-studied  bacterial  poison,  a  discovery  made  by 
Roux  in  collaboration  with  Yersin,  it  has  been  recognised  that  mice 
and  rats  tolerate  large  quantities  of  diphtheria  cultures  or  of  their 
filtered  products.  A  rat  resists  a  dose  of  the  diphtheria  poison 
capable  of  killing  several  rabbits.  To  explain  this  great  natural 
immunity  it  was  suggested  that  the  antitoxic  property  of  the 
body  fluids  could  be  called  in.  It  was  supposed  that  the  rat's  blood 
was,  by  its  very  nature,  endowed  with  the  power  of  neutralising  the 

1  "  Le  venin  des  serpents,"  Paris,  1896,  p.  43. 

2  The  temporary  immunity  of  the  marmot  (amongst  mammals)  against  tetanus 
toxin  must  be  considered  separately.    According  to  Billinger  and  Donitz  the  marmot 
is  insusceptible  to  this  poison  during  its  winter  sleep.    But  once  it  is  awakened  it 
readily  contracts  tetanus.     H.  Meyer,  Halsey  and  Ransom  have  observed  the  same 
fact  in  hibernating  bats  that  have  been  waked  up.     In  these  cases  the  immunity  is 
dependent  on  the  low  temperature  which  approximates  these  examples  to  that  of  the 
natural  immunity  of  the  frog  against  the  same  toxin. 

22—2 


340  Chapter  XI 

toxin  of  diphtheria.  But,  as  in  the  tetanus  of  fowls,  it  was  not  long 
[357]  before  facts  rendered  this  hypothesis  untenable.  Kuprianow1  studied 
this  question  under  the  direction  of  Loeffler  and  gave  an  account 
of  the  results  of  his  experiments,  which  proved  that  the  blood  of  the 
sewer  rat,  which  is  very  refractory  against  diphtheria,  contains  no 
substance  that  will  neutralise  the  morbific  action  of  diphtheria  toxin 
on  susceptible  animals,  especially  the  guinea-pig. 

It  was  necessary  to  seek  some  other  explanation,  and  the  idea 
that  the  immunity  of  the  rat  depends  on  the  insusceptibility  of  its 
living  cells  to  the  diphtheria  poison  was  seized  upon.  The  experi- 
ments carried  out  by  Roux  and  Borrel2  demonstrated  the  incorrect- 
ness of  this  hypothesis.  The  immunity  of  rats  to  subcutaneous  or 
intra-peritoneal  injection  of  diphtheria  toxin  is  very  marked.  But 
a  very  small  dose  (O'l  c.c.)  of  this  poison,  introduced  directly  into 
the  cerebral  substance  of  the  rat,  produces  a  complete  paralysis,  which 
lasts  for  several  days,  and  ends  in  the  death  of  the  animal.  Roux 
and  Borrel  conclude  from  this  "  that  the  brain  of  the  rat  is  specially 
sensitive  to  the  action  of  the  diphtheria  poison,  and  that  as  this 
animal  does  not  die  as  the  result  of  the  injection  of  large  quantities 
of  toxin  into  the  subcutaneous  tissue,  it  is  because  the  toxin  does 
not  reach  the  brain."  These  authors  have  pointed  out  analogous 
facts  in  connection  with  other  examples  of  natural  immunity.  The 
rabbit,  which  withstands  a  hypodermic  injection  of  30  centigrammes 
of  chlorhydrate  of  morphia,  is  killed  by  1  milligramme  only  of  this 
salt,  introduced  directly  into  the  brain.  Here,  again,  neither  the 
cellular  insusceptibility  nor  the  antitoxic  property  of  the  blood  (no 
"  antialkaloidal "  power  could  ever  be  demonstrated)  can  explain  the 
immunity,  which  appears  to  be  due  rather  to  the  factor  which  arrests 
the  poison  on  its  way  to  the  nerve  centres. 

In  spite  of  the  insufficiency  of  our  knowledge  as  regards  natural 
immunity  against  soluble  poisons  we  are  quite  justified  in  affirming 
that  this  category  of  phenomena  comes  mainly  into  the  domain  of 
the  cells.  The  body  fluids  of  animals  which  exhibit  this  immunity 
have  been  found  to  be  antitoxic  in  a  few  species  only  (scorpion, 
snake,  hedgehog,  mongoose).  And  for  the  majority  of  these  it  is 
possible  to  invoke  special  causes,  such  as  the  internal  secretion  of 
snake  and  scorpion  venoms  by  the  glands  which  manufacture  them, 
or  the  acquisition  of  an  antitoxic  power  during  life  resulting  from 

1  Centralbl.f.  Bakteriol  u.  Parasitenk.,  Jena,  1894,  Bd.  xvi,  S.  415. 

2  Ann.  de  I'Inst.  Pasteur,  Paris,  1898,  t.  xu,  p.  225. 


Natural  immunity  against  toxins  341 

•wounds  or  from  the  absorption  of  venomous  food.  The  theory  of  [358] 
the  insusceptibility  of  the  cells  of  animals  naturally  refractory  to 
toxins  must  also  be  rejected  ;  it  is  incompatible  with  well-established 
facts.  Nothing  remains,  then,  but  to  assume  that  the  formed 
elements  are  the  principal  factors  in  this  natural  immunity,  and  that 
they  interpose  to  prevent  the  passage  of  the  poisons  towards  the 
very  susceptible  nerve  cells. 


[359]  CHAPTER  XII 

ARTIFICIAL  IMMUNITY  AGAINST  TOXINS 

Adaptation  to  poisons. — Artificial  immunity  against  bacterial  and  vegetable  toxins 
and  against  snake  venom. — Principal  methods  of  immunisation. — Immunisation 
by  toxins  and  toxoids. — Inoculation  against  diphtheria  toxin. — Phenomena 
produced  in  the  course  of  vaccination  against  toxins. — Rise  of  temperature. — 
Leucocytosis. — Development  of  antitoxic  power. — Properties  of  antitoxins. — 
Mode  of  action  of  antitoxins. — Action  of  antitoxins  in  vitro. — Their  action  in 
the  organism. — Influence  of  living  elements  on  the  combination  of  antitoxin 
with  toxin. — Antitoxic  action  of  non-specific  serums,  of  normal  serums  and  of 
broth. — Immunity  against  toxins  is  not  in  direct  ratio  to  the  amount  of  anti- 
toxins in  the  body  fluids. — Hypersensitiveness  of  an  animal  treated  with  toxin. — 
Diminution  of  the  susceptibility  of  the  organism  immunised  against  toxins. 

Hypotheses  as  to  the  nature  and  origin  of  antitoxins. — Hypothesis  of  the  transforma- 
tion of  toxins  into  antitoxins. — Hypothesis  of  receptors  detached  from  cells  as 
the  source  of  antitoxins. — Hypothesis  of  the  nervous  origin  of  tetanus  antitoxin. 
— Fixation  of  tetanus  toxin  by  the  substance  of  the  nerve  centres. — The  relations 
between  saponin  and  cholesterin. — Anti-arsenic  serum. — Part  played  by  phago- 
cytes in  the  struggle  of  the  animal  against  poisons. — Probable  part  played  by 
phagocytes  in  the  production  of  antitoxins. 

ALTHOUGH  scientific  men  succeeded  only  a  little  more  than  ten 
years  ago  in  vaccinating  against  poisons  by  artificial  methods,  savage 
races  and  ancient  peoples  at  a  very  remote  period  undoubtedly  pos- 
sessed methods  of  counteracting  the  effects  of  certain  venomous 
substances.  The  frequent  observation  of  cases  in  which  doses  of 
poisons,  insufficient  to  cause  death,  brought  about  a  more  or  less 
durable  resistant  condition,  must  result  in  the  elaboration  of  artificial 
means  of  preventing  the  intoxications. 

Von  Behring1  points  out  that  analogous  facts  must  have  been 
known  to  the  physicians  of  ancient  times ;  and  it  is  in  such  know- 
ledge that  we  must  look  for  the  source  of  the  dogma  put  forward 
by  Hippocrates,  that  the  factor  which  produces  a  disease  is  also 
capable  of  curing  it. 

1  "  Allgemeine  Therapie  der  Infectionskrankheiten,"  Berlin  u.  Wien,  1899,  S.  982. 


Artificial  immunity  against  toxins  343 

To  Piiny  we  are  indebted  for  the  now  well-known  story,  that 
Mithridates  of  Pontus  possessed  the  means   of  protecting  himself 
against  various  poisons   by  a  process  of  adaptation,  and,  amongst  [360] 
others,  by  the  use  of  the  blood  of  Pontine  ducks  to  which  he  had 
given  poisons  by  the  mouth. 

The  adaptation  of  horses  and  of  the  Highlanders  of  Styria  to  arsenic, 
as  well  as  that  of  the  many  morphinomaniacs  to  morphia,  is  known 
to  everybody.  A  man,  habituated  to  morphia,  is  able  to  consume 
daily  a  dose  several  times  the  fatal  one ;  indeed,  cases  have  been 
known  of  people  acquiring  the  power  of  consuming  two,  and  even 
three,  grammes  of  morphia  per  diem.  Man  may  acquire  an  adapta- 
tion to  toxic  substances  of  the  most  diverse  character,  such  as 
arsenic,  alcohol,  morphia,  nicotine,  etc. 

Even  when  we  had  obtained  much  information  concerning  acquired 
immunity  against  micro-organisms  we  still  knew  nothing  of  the 
mechanism  of  such  adaptation,  or  as  to  the  possibility  of  acquiring  a 
special  immunity  against  bacterial  poisons.  Charrin  and  Gamaleia's 
discovery  that  animals  vaccinated  against  a  micro-organism  are  just 
as  susceptible  to  its  toxic  products  as  normal  animals,  led  Bouchard1, 
in  whose  laboratory  it  was  made,  to  say  that  the  idea  of  the  adapta- 
tion of  cells  to  bacterial  poisons  must  be  dropped.  He  developed 
this  thesis  at  the  International  Congress  at  Berlin  in  1890,  and 
formulated  it  as  follows  :  "  When  we  inject  a  healthy  animal  and 
a  vaccinated  one  with  the  soluble  products  of  the  micro-organism 
which  has  been  used  for  the  vaccination,  the  dose  required  to  kill 
each  animal  is  exactly  the  same.  Let  us  not  speak,  then,  of  the 
training  of  the  leucocytes,  and  of  the  adaptation  of  the  nerve  cells 
to  bacterial  poisons  :  it  is  pure  rhetoric."  At  this  time  we  had  only 
just  commenced  to  acquire  exact  knowledge  concerning  the  toxins 
of  micro-organisms.  For  a  considerable  period  they  were  sought 
for  amongst  the  ptomains,  very  stable  substances  allied  to  the 
alkaloids ;  here,  however,  we  were  working  in  a  wrong  direction.  It  was 
not  until  the  classic  researches  of  Roux  and  Yersin2  on  diphtheria 
toxin,  published  in  1888  and  1889,  that  the  true  nature  of  bacterial 
poisons  was  revealed.  It  was  found  that  we  were  not  dealing  with 
ptomains,  but  with  soluble  ferments,  substances  of  indeterminate 
chemical  composition,  allied  to  the  albuminoids,  and,  like  them, 

1  "Essai  d'une  theorie  de  1'infection,"  Berlin,  1890;  "Les  microbes  pathogenes," 
Paris,  1892,  p.  33. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1888,  t.  n,  p.  629;  1899,  t  in,  p.  273. 


344  Chapter  XII 

[361]  unstable.  The  methods  adopted  by  Roux  and  Yersin  in  their  study 
of  diphtheria  toxin  enabled  other  investigators  to  discover  the  analo- 
gous toxins  of  several  other  bacteria.  Knud  Faber1  and  Brieger  and 
Frankel2  soon  succeeded  in  separating  the  toxin  from  the  tetanus 
bacillus,  a  toxin  capable  of  producing  in  animals  tetanic  contractions 
as  typical  as  those  obtained  with  cultures  of  the  tetanus  bacillus. 

These  investigations  inaugurated  a  new  era  in  microbiology  and 
enabled  us  to  attack  the  problem  of  acquired  immunity  against 
bacterial  toxins  scientifically.  Within  a  few  mouths  of  the  declaration 
made  by  Bouchard  at  the  Berlin  Congress,  there  appeared,  almost 
simultaneously,  the  earliest  publications  on  the  possibility  of  vaccinat- 
ing laboratory  animals  against  the  toxins  of  diphtheria  and  tetanus 
by  artificial  methods.  Immediately  after  the  discovery  of  these 
poisons,  the  attempt  was  made  to  immunise  various  species  of  animals 
against  them,  but  here  very  great  difficulties  were  met  with;  the 
animals,  after  receiving  increasing  doses  of  toxin,  became  thin 
and  ultimately  died.  It  occurred  to  Frankel3  that  the  toxic 
action  of  the  diphtheria  poison  might  be  weakened  by  subjecting 
it  to  a  temperature  of  60°  C.  Independently,  von  Behring  and 
Kitasato4  used  chemical  substances,  especially  iodine  trichloride,  to 
attenuate  the  action  of  the  tetanus  and  diphtheria  toxins.  The 
animals  which  resisted  these  modified  poisons  were  found  to  be 
capable  of  tolerating  gradually  increasing  doses  of  unaltered  and 
very  active  toxins.  By  the  use  of  these  methods  it  was  found 
possible  to  obtain  a  definite  and  lasting  immunity  against  these 
microbial  products. 

The  discovery  of  the  possibility  of  vaccinating  against  bacterial 
toxins  was  soon  followed  by  the  demonstration  of  the  antitoxic  power 
of  the  blood  of  animals  that  had  acquired  such  artificial  immunity 
against  these  poisons.  Everyone  knows  of  and  appreciates  von  Behring 
and  Kitasato's  great  discovery.  It  opened  up  a  new  and  fruitful  field 
of  research  from  most  diverse  points  of  view.  Ehrlich5  was  able  to 

[362]  apply  it  to  the  vaccination  of  animals  against  the  vegetable  poisons 
ricin,  abrin  and  robin,  and  thus  to  establish  rigorous  methods  of  im- 
munisation and  to  obtain  very  important  results  concerning  immunity 
against  toxins  in  general.  He  also  succeeded  in  demonstrating  that 

1  Berl  klin.  Wchnschr.,  1890,  S.  717. 

2  Berl.  klin.  Wchnschr.,  1890,  No.  11. 

3  Berl  klin.  Wchnschr.,  1890,  No.  49. 

4  Deutsche  med.  Wchnschr.,  Leipzig,  1890,  SS.  1145,  1245. 

5  Deutsche  med.  Wchnschr.,  Leipzig,  1891,  SS.  976,  1218. 


Artificial  immunity  against  toxins  345 

animals  vaccinated  against  these  vegetable  poisons,  which,  by  their 
nature,  approximate  to  the  microbial  toxins,  develop  in  their  blood 
a  most  marked  antitoxic  property. 

Some  years  later,  the  discovery  of  antitoxins  was  extended  to 
snake  venoms,  poisons  of  animal  origin  which,  like  the  vegetable 
poisons  studied  by  Ehrlich,  present  a  chemical  composition  analogous 
to  that  of  the  microbial  toxins.  Phisalix  and  Bertrand1  and  Calmette2, 
working  independently,  discovered  methods  of  vaccination  against 
snake  venom  and  were  able  to  demonstrate  the  existence  of  an 
antitoxic  power  of  the  blood  in  immunised  animals. 

The  works  above  briefly  referred  to  gave  us  the  fundamental  basis 
of  our  present  knowledge  on  acquired  immunity  against  toxins. 

It  would  be  very  interesting  to  be  able  to  determine  whether  the 
lower  animals  can  be  vaccinated  against  the  toxic  substances  to  which 
they  are  susceptible.  Unfortunately  in  the  study  of  this  problem 
we  encounter  very  great  difficulties.  Making  use  of  various  methods 
I  have  often  tried  to  solve  it.  The  crayfish  is  susceptible  to  snake 
venom  and  to  the  ichthyotoxin  of  eel's  serum,  and  I  have  tried  at 
various  times  to  vaccinate  it  against  these  poisons.  The  results, 
however,  were  so  inconstant  and  even  contradictory  that  I  was 
unable  to  draw  any  definite  conclusion  from  them. 

It  is,  indeed,  very  difficult  to  vaccinate  the  lower  vertebrata  against 
poisons.  Several  attempts  have  been  made  in  my  laboratory  to 
immunise  frogs  against  tetanus  toxin,  but  without  success.  Calmette 
and  Delearde3  obtained  the  best  results  with  abrin.  They  succeeded 
in  vaccinating  frogs — which  are  not  very  susceptible  to  this  vegetable 
toxin,  though  they  are  far  from  presenting  a  real  natural  immunity — 
against  doses  which  are  absolutely  fatal  for  the  control  animals.  These 
observers,  however,  had  to  proceed  very  cautiously,  and  they  allowed 
a  very  long  interval  between  each  injection  of  abrin.  The  blood  of 
their  vaccinated  frogs  not  only  did  not  prove  to  be  antitoxic  against  [363] 
abrin,  when  injected  into  mice,  but  for  long  retained  sufficient  of  this 
toxin  to  poison  normal  mice.  This  experiment  certainly  tells  against 
the  hypothesis  that  the  acquired  immunity  of  frogs  is  due  to  the 
development  of  a  specific  antitoxic  power  in  their  body  fluids,  but  it 
does  not  settle  the  question  definitely  since  it  may  be  objected  that 

1  Compt.  rend.  Soc.  de  biol.,  Paris,  1894,  p.  111. 

2  Compt.  rend.  Sue.  de  biol.,  Paris,  1894,  pp.  120,  204.    [Cf.  also  Fraser,  Brit.  Med. 
Journ.,  London,  1895,  Vol.  i,  p.  1309  and  n,  p.  416;  Nature,  London,  1896,  Vol.  mi, 
p.  571.] 

3  Ann.  de  VInst.  Pasteur,  Paris,  1896,  t.  x,  p.  683. 


346  Chapter  XII 

the  blood,  whilst  toxic  for  mice,  might,  still,  be  antitoxic  for  the  frog. 
The  antitoxin  of  this  blood  might  merely  be  incapable  of  neutralising 
all  the  abrin  present.  Fresh  investigations,  then,  are  necessary. 

Even  in  the  higher  vertebrata,  it  is  often  very  difficult  to  obtain 
a  real  vaccination  against  the  various  toxins.  In  the  small  mammals, 
which  exhibit  a  great  susceptibility  to  these  poisons,  it  is  specially 
difficult  to  obtain  an  artificial  immunity.  As  Vaillard  and  von  Behring 
have  demonstrated,  it  is  possible  to  vaccinate  such  animals  by  means 
of  gradually  increasing  doses  of  unmodified  toxins,  but  this  method 
demands  much  time,  is  often  dangerous,  and  hence  is  not  very 
practical.  Poisons  that  act  through  the  alimentary  canal  are  the 
most  serviceable  for  vaccination,  as  has  been  demonstrated  by 
Ehrlich.  This  investigator  had  to  abandon  the  vaccination  of  mice 
by  means  of  subcutaneous  injections  of  ricin  on  account  of  the  slough- 
ing set  up  at  the  point  of  inoculation.  He  then  had  recourse  to 
vaccination  by  way  of  the  mouth,  which  gave  very  good  results,  not 
only  with  ricin  but  also  with  abrin.  This  mode  of  vaccination,  how- 
ever, is  applicable  to  a  small  number  of  poisons  only. 

We  can  also  vaccinate  mammals,  even  laboratory  rodents,  such  as 
rabbits  and  guinea-pigs,  by  means  of  unmodified  snake  venom,  but 
this  method  is  a  very  delicate  one  and  must  be  carefully  watched. 
It  is  necessary  to  begin  with  very  small  doses  of  venom,  continue 
them  for  some  time,  and  increase  the  amount  of  venom  injected  very 
slowly.  Calmette1  modified  this  method  by  inserting,  below  the  skin 
and  leaving  it  there,  a  piece  of  chalk  impregnated  with  small  quantities 
of  venom  and  surrounded  by  collodion  through  which  the  venom 
diffuses  very  slowly  and  continuously. 

[364]  Large  mammals,  sheep,  oxen  and  horses,  can  be  more  easily 
vaccinated  by  means  of  unmodified  toxins,  but  they  also  require 
to  be  treated  with  very  special  precaution.  Salomonsen  and  Madsen2 
have  given  the  history  of  their  horse,  immunised  with  diphtheria 
toxin.  Into  a  mare  weighing  665  kilos  they  were  able  to  inject 
at  the  commencement  only  1  c.c.  of  this  toxin,  and  the  dose  had  to 
be  increased  very  carefully. 

In  the  presence  of  all  these  difficulties  in  the  use  of  unmodified 
toxins  for  vaccination,  a  different  method  is  now  generally  adopted 
in  the  immunisation  of  animals,  small  or  large,  for  the  purpose  of 
scientific  research  or  for  the  preparation  of  toxins  on  a  commercial 

1  "Le  venin  des  serpents,"  Paris,  1896,  p.  54. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1897,  t.  xi,  p.  316. 


Artificial  immunity  against  toxins  347 

scale.  Vaccination  is  commenced  with  toxins  modified  by  heat  or  by 
chemical  substances.  The  diphtheria  and  tetanus  toxins,  those  most 
employed  in  the  serotherapeutic  industry,  are  subjected  to  various 
degrees  of  heat.  Frankel1  was  the  first  to  make  use  of  this  method 
for  vaccination  against  diphtheria,  and  Vaillard2  for  vaccination 
against  tetanus.  It  consists  in  introducing  large  doses  of  filtered 
cultures,  heated  to  progressively  lower  degrees  of  temperature,  60°, 
55°,  50°  C.,  and  then  giving  gradually  increasing  quantities  of  filtered 
cultures  whose  toxicity  is  unaltered.  This  method  is  very  convenient 
for  small  animals,  but  for  large  mammals  it  is  greatly  simplified  by  in- 
jecting for  a  certain  period  toxins  heated  to  60°  C.,  and,  later,  replacing 
these  by  unmodified  toxin. 

Phisalix  and  Bertrand3  applied  an  analogous  method  to  the 
vaccination  of  the  guinea-pig  against  the  venom  of  the  viper.  This 
poison,  which  resists  much  higher  temperatures  than  do  the  tetanus 
and  diphtheria  toxins,  received  a  preliminary  heating  to  80°  C.  in 
order  that  it  might  be  inoculated  without  danger  into  small  animals. 
Under  these  conditions  it  confers  a  certain  immunity,  but  even  when 
heated  to  80°  C.  it,  in  many  cases,  still  remains  sufficiently  active  to 
produce  fatal  results.  For  this  reason,  in  the  vaccination  of  animals 
for  the  preparation  of  antivenomous  serum  on  a  large  scale,  Calmette 
had  recourse  to  another  method,  that  of  attenuating  the  venom  by  [365] 
means  of  chemical  substances. 

Von  Beh ring  and  Kitasato4  were  the  first  to  make  use  of  iodine 
trichloride  in  the  vaccination  of  animals  against  the  toxins  of  tetanus 
and  diphtheria.  In  their  early  experiments  this  substance  was  injected 
before  the  toxins  were  introduced.  Later,  the  mixture  was  made 
in  vitro  and  then  injected  into  the  animals.  Roux  devised  another 
method  which  had  the  advantage  of  being  simple,  certain,  and  easily 
employed,  for  which  reason  it  was  soon  introduced  into  commercial 
and  scientific  practice.  It  consists  in  the  injection  of  mixtures  of  the 
tetanus  or  diphtheria  toxins  with  LugoFs  iodo-ioduretted  solution. 
The  iodine,  in  small  doses,  instantly  neutralises  or  modifies  these 
poisons  and  is  itself  borne  well,  even  by  small  animals.  By  employing 
progressively  increasing  doses  of  these  mixtures,  in  which  the  amount 

1  Berl.  klin.  Wchnschr.,  1890,  No.  49. 

2  Ann.  de  llnst.  Pasteur,  Paris,  1892,  t.  vi,  p.  225. 

3  Compt.  rend.  Acad.  d.  sc.y  Paris,  1894,  t.  cvm,  p.  288 ;  Compt.  rend.  Soc.  de 
liol,  Paris,  1894,  p.  111. 

4  Deutsche  med.  Wchnschr.,  Leipzig,  1890,  SS.  1145,  1245. 


348  Chapter  XII 

of  iodised  solution  becomes  smaller  and  smaller  compared  with  that 
of  the  toxin,  we  are  able,  without  difficulty,  to  vaccinate  the  most 
susceptible  animals  and  enable  them  to  withstand  considerable  doses 
of  the  pure  toxin.  By  this  method  it  is  possible  to  immunise  guinea- 
pigs  against  the  most  active  tetanus  toxin.  The  method  serves  equally 
well  for  the  preparation  of  horses  for  injections  of  unmodified  toxins. 
For  a  longer  or  shorter  time  (according  to  the  susceptibility  of  the 
horse)  toxins  which  are  mixed  with  Lugol's  iodised  water  are 
injected.  Having  made  sure  of  the  resistance  of  the  horse,  larger 
and  larger  quantities  of  pure,  unmodified  toxin  may  be  introduced 
with  impunity. 

For  the  immunisation  of  mammals  of  all  sizes  (guinea-pigs,  rabbits, 
dogs,  horses)  against  snake  venom,  Calmette,  in  his  work  at  Lille,  also 
makes  use  of  venom  modified  by  chemical  substances,  but  his  method 
differs  from  those  we  have  just  described.  During  several  weeks  he 
injects  increasing  quantities  of  venom,  mixed  with  decreasing  quantities 
of  a  solution  of  1 : 60  of  hypochlorite  of  lime.  After  this  treatment  the 
animals  become  capable  of  tolerating  fatal  doses  of  unmodified  venom 
and  can  be  injected  with  larger  and  larger  doses. 

In  recent  years  a  method  of  vaccinating  horses  against  certain 
microbial  toxins,  and  especially  against  the  diphtheria  toxin,  by  means 
of  mixtures  of  toxin  and  antitoxic  serum,  or  with  these  two  products 
successively,  has  been  introduced.  Babes1  was  the  first  to  extol 
[366]  this  method  as  the  best  for  obtaining  a  high  and  durable  immuni- 
sation. Afterwards,  several  other  observers,  amongst  whom  I  may 
cite  Pawlowsky  and  Maksutow2,  Palmirsky,  and  especially  Nikanoroff3, 
took  up  this  question,  and  communicated  very  encouraging  results. 
Von  Behring4  also  found  it  very  useful  in  certain  cases.  Thus,  for 
the  vaccination  of  guinea-pigs  against  tetanus  toxin,  he  recommends 
the  injection  of  a  mixture  containing  antitoxin  and  an  unneutralised 
excess  of  toxin.  Under  these  conditions  he  easily  succeeds  in  im- 
munising these  small  animals  in  cases  where  all  other  methods  fail. 
As  a  general  method  of  vaccination  against  toxins,  however,  this 
method  has  not  fulfilled  its  promise,  and  Roux,  who  tried  it  several 
times,  was  not  at  all  satisfied  with  it. 

1  Bull.  Acad.  de  med.,  Paris,  1895,  t.  xxxiv,  p.  216. 

2  Ztschr.f.  Hyg.,  Leipzig,  1896,  Bd.  xxi,  S.  485. 

"•On  the  preparation  of  a  potent  antidiphtheria  serum,"  St-Petersbourg,  1897 
(in  Russian)  [cf.  Berl  klin.  Wchnschr.,  1897,  S.  720]. 

4  "  ADgemeine  Therapie  der  Infectionskrankheiten,"  Berlin  u.  Wien,  1899.  S.  1093. 


Artificial  immunity  against  toxins  349 

This  method  of  immunisation  by  mixtures  of  toxin  and  antitoxin 
is  often  spoken  of  as  the  method  of  vaccination  by  toxones.  This 
name,  "toxone,"  was  first  applied  by  Ehrlich1  to  a  product  developed 
by  the  diphtheria  bacillus  in  culture  media,  a  product  less  and 
differently  toxic  than  is  the  true  diphtheria  toxin,  yet  capable  of 
neutralising  antitoxin.  The  idea  of  toxones  presented  itself  to 
Ehrlich  in  connection  with  a  fundamental  fact  noted  by  him,  namely, 
that  when  to  a  non-toxic  mixture  of  diphtheria  toxin  and  antitoxin 
there  is  added  one  and  even  several  lethal  doses  of  the  former,  the 
animal  is  not  affected.  To  make  it  succumb  to  intoxication  it  is 
sometimes  necessary  to  add  more  than  20  lethal  doses  of  toxin.  To 
explain  this  paradoxical  result,  Ehrlich  formulated  the  hypothesis 
that,  in  the  soluble  products  of  the  diphtheria  bacillus  there  exist 
two  poisons  :  (1)  the  true  toxin  which  exhibits  a  very  strong  affinity 
for  antitoxin,  and  (2)  the  toxone  which  possesses  less  avidity  for  this 
antibody.  When  to  an  inactive  mixture  of  the  products  of  diphtheria 
bacilli  and  of  antitoxin,  there  is  added  a  fresh  quantity  of  these  same 
products,  the  added  toxin,  owing  to  its  greater  affinity,  replaces  the 
toxone  of  the  previous  combination.  In  the  mixture  to  which  is 
added  one  or  several  lethal  doses  of  diphtheria  poison,  the  toxone  [367] 
only  is  found  free,  all  the  toxin  being  combined  writh  the  antitoxin, 
and,  as  the  toxone  is  only  feebly  toxic,  the  animal  resists  without 
suffering  any  serious  illness. 

Madsen2  adopted  the  theory  of  the  diphtheria  toxone,  and  affirmed 
that  this  substance  poisons  but  slowly,  produces  neither  early  nervous 
symptoms  nor  loss  of  hair,  but  excites  slight  oedema  at  the  point  of 
inoculation  and  late  paralyses.  Susceptible  animals  may  olie  from 
toxones,  but  very  much  later  than  as  the  result  of  poisoning  by  the 
toxins. 

Ehrlich's  pupils  have  extended  the  theory  of  toxones  to  other 
bacterial  poisons.  Thus  Madsen3  has  described  a  similar  toxone  in 
tetanus  poison — the  tetanolysin  of  Ehrlich — which  dissolves  the  red 
blood  corpuscles,  and  Neisser  and  Wechsberg4  refer  to  a  toxone  in 
the  poison  produced  by  the  staphylococcus. 

Ehrlich  also  describes  toxoids  as  occurring  in  diphtheria  poison. 
The  toxone,  he  maintains,  is  a  product  of  the  diphtheria  bacillus 

1  Deutsche  med.  Wchnschr.,  Leipzig,  1898,  S.  597. 

2  Ztschr.f.  Hyg.,  Leipzig,  1897,  Bd.  xxiv,  S.  425. 

3  Ann.  de  Flnst.  Pasteur,  Paris,  1899,  t  xra,  pp.  568,  801. 

4  Ztschr.f.  Hyg.,  Leipzig,  1901,  Bd.  xxxvi,  S.  325. 


350  Chapter  XII 

itself,  but  the  toxoids  (protoxoids  and  syntoxoids)  represent  the 
toxin  modified  without  further  aid  from  the  bacillus.  The  toxoids, 
though  not  toxic,  retain  all  their  avidity  for  antitoxin.  According  to 
Ehrlich's  conception,  the  molecule  of  toxin,  under  the  influence  of 
various  factors,  readily  loses  its  toxic  or  toxophore  group,  capable  of 
poisoning  the  animal,  whilst  still  retaining  its  haptophore  group,  the 
group  that  combines  with  the  antitoxin.  The  toxoids  then  would 
represent  this  haptophore  group  of  the  diphtheria  toxin.  Without 
being  injurious  to  animals,  the  toxoids  are  capable  of  neutralising 
the  antitoxin  and  of  setting  up  in  the  animal  the  formation  of  this 
antibody.  In  the  experiments  carried  out  by  the  method  of  Babes 
and  of  the  Russian  authors  we  have  just  mentioned,  there  would  be, 
according  to  the  view  held  by  Ehrlich  and  his  school,  an  immuni- 
sation by  the  toxoids. 

The  toxones,  however,  are  also  capable  of  vaccinating  against  the 
toxin  and  the  toxone  and  of  giving  rise  to  the  production  of  a 
diphtheria  antitoxin,  active  against  these  two  poisons.  This  is  what 
[368]  is  affirmed  by  Madsen1  and  by  Dreyer2,  according  to  a  communication 
made  by  the  latter  to  the  International  Congress  of  Medicine  held 
at  Paris. 

By  means  of  the  various  methods  briefly  described  above,  is 
obtained  a  real  acquired  immunity  against  the  various  bacterial 
and  vegetable  poisons  and  the  venoms.  On  the  other  hand,  with 
the  methods  of  vaccination  mentioned  in  the  eighth  chapter,  which 
confer  a  substantial  immunity  against  micro-organisms,  we  cannot 
demonstrate,  in  the  vaccinated  animals,  a  resistance  against  the 
corresponding  toxins  greater  than  in  the  unvaccinated  control 
animals.  The  animals,  so  thoroughly  vaccinated  against  certain 
micro-organisms  that  they  withstood  enormous  doses  of  culture, 
did  not  become  capable  of  resisting  the  minimal  lethal  dose  of 
the  poison.  We  are  led  to  conclude,  therefore,  that  immunity  can 
only  be  obtained  against  certain  of  the  toxins.  For  this  reason  we 
must  regard  the  attempt  made  by  von  Behring  to  obtain  a  real 
immunisation  against  the  toxin  of  cholera  as  an  important  forward 
step.  Before  von  Behring's  attempt,  various  species  of  animals  had 
been  frequently  and  very  substantially  vaccinated  against  the  cholera 

1  Compt.  rend,  du  Congres  internat.  de  med.  de  Paris  (Section  de  bacteriologie 
ct  parasitologie),  1901,  p.  40. 

2  Compt.  rend,  du  Congres  internat.  de  med.  de  Paris  (Section  de  bacteriologie 
et  parasitologie),  1901,  p.  45  ;  Ztschr.  f.  Hyg.,  Leipzig,  1901,  Bd.  xxxvn,  S.  250. 


Artificial  Immunity  against  toxins  351 

vibrio,  but  these  animals,  even  when  most  thoroughly  vaccinated,  were 
completely  non-resistant  to  the  cholera  toxin.  Von  Behring  suggested 
to  his  pupil  Ransom1  the  idea  of  immunising  guinea-pigs,  not  with 
microbial  cultures  living  or  dead,  as  had  usually  been  done  previously, 
but  exclusively  with  the  fluids  of  the  cultures,  deprived  of  the 
vibrios  by  filtration.  In  order,  however,  to  attain  the  desired  object, 
it  was  necessary  to  prepare  fluids  sufficiently  active  to  poison  the 
unvaccinated  control  guinea-pigs  with  certainty.  The  results  of  these 
investigations  confirmed  his  anticipation,  and  Ransom  soon  found 
himself  in  possession  of  guinea-pigs  well  vaccinated  against  the 
cholera  poison.  He  was  mistaken,  however,  in  supposing  that,  in  all 
cases  of  immunity  acquired  against  Koch's  vibrio,  we  have  to  do,  in 
the  main,  with  a  purely  antitoxic  immunity.  An  investigation  carried 
out  in  the  Pasteur  Institute2,  whilst  confirming  the  facts  discovered 
by  Ransom,  lead  to  different  results  as  regards  their  interpretation. 
It  was  demonstrated  that  the  immunity  against  the  vibrio  is  in  no  [369] 
way  founded  on  a  resistance  against  its  toxin  and  that  we  have  to 
do  with  two  very  different  acquired  immunities.  The  vaccination 
obtained  with  the  bodies  of  the  micro-organisms  induced  a  refractory 
condition  against  infection  by  the  living  vibrio,  but  not  the  slightest 
resistance  against  the  toxin.  The  immunity,  on  the  other  hand, 
which  is  conferred  by  the  injection  of  soluble  products,  deprived  of 
the  micro-organisms,  is  effective  not  only  against  the  toxin  of  cholera, 
but  also  against  infection  by  the  vibrio.  When  an  animal  is  vac- 
cinated with  cultures,  or  even  with  the  bodies  only  of  the  vibrios, 
cholera  toxin  is  introduced,  but  the  toxin,  under  these  conditions,  is 
incapable  of  setting  up  antitoxic  immunity.  It  would  appear  that 
the  presence  of  the  vibrios  may  constitute  some  obstacle  to  the  pro- 
duction of  this  immunity. 

Soon  afterwards,  Wassermann3  pointed  out  that  the  same  rule 
applies  in  the  case  of  the  Bacillus  pyocyaneus.  With  whole  cultures 
of  this  bacillus  he  obtained  in  guinea-pigs  an  immunity  exclusively 
against  infection,  whilst  with  cultures  in  a  fluid  medium,  deprived  of  the 
bacilli,  he  was  able  to  vaccinate  his  animals  both  against  the  pyocy- 
anic  toxin  and  against  the  infective  peritonitis  produced  by  the  living 
micro-organism.  The  same  double  immunity  could  also  be  obtained 


1  Deutsche  med.  Wchnschr.,  Leipzig,  1895,  S.  457. 

2  Ann.  de  Flnst.  Pasteur,  Paris,  1896,  t.  x,  p.  257. 

3  Ztschr.f.  Hyg.,  Leipzig,  1896,  Bd.  XXH,  S.  312. 


352  Chapter  XII 

in  laboratory  animals  against  the  typhoid  bacillus  and  several  other 
bacteria. 

When  animals  were  subjected  to  different  methods  of  vaccination 
against  toxins,  the  manifestation  of  certain  phenomena  more  or  less 
constant  was  observed ;  amongst  these  must  be  pointed  out  especially 
the  rise  of  temperature,  a  local  reaction  and  certain  modifications  in 
the  body  fluids. 

Fever  is  a  very  general  symptom  in  the  course  of  the  vaccination 
of  mammals.  A  rise  of  temperature  is  almost  always  observed  as  a 
result  of  the  injection  of  toxins.  It  is  very  variable,  both  as  regards 
duration  and  intensity,  and  cannot  serve  as  an  indicator  of  the  result 
of  the  vaccination.  In  this  respect,  such  great  differences  have  been 
observed  that  the  attempt  to  establish  any  general  laws  has  had  to  be 
abandoned. 

Local  reaction  is  also  a  phenomenon  which  is  very  frequently 
observed  during  vaccination  ;  to  this  von  Behring1  paid  great  atten- 
tion. He  and  his  collaborators  found  that  normal  horses  when 
[370]  injected  subcutaneously  with  small  or  large  doses  of  tetanus  toxin 
did  not  present  any  exudation  at  the  seat  of  inoculation.  The  horses 
which  died  as  the  result  of  a  tetanus  intoxication  and  those  which 
got  better  behaved  from  this  point  of  view  in  much  the  same  fashion. 
In  horses,  however,  which  are  being  vaccinated  and  which  are 
periodically  subjected  to  gradually  increasing  doses  of  toxin,  tume- 
faction at  the  seat  of  injection  is  never  absent.  Von  Behring 
attributes  this  difference  to  the  primordial  insusceptibility  of  the 
living  elements  which  govern  exudation  in  the  subcutaneous  tissue 
to  tetanus  poison.  It  is  only  during  the  process  of  vaccination  that 
these  cells  become  susceptible  and  capable  of  manifesting  a  visible 
reaction.  I  consider  that  this  difference  is  due  more  probably  to  a 
change  in  the  chemiotaxis  of  the  various  elements  which  contribute 
to  the  inflammatory  exudation  reaction,  from  a  negative  to  positive 
type.  The  cells  do  not  react  at  the  commencement,  not  because  they 
are  not  susceptible  to  the  toxin,  but  rather  because  their  suscepti- 
bility is  too  great.  During  the  course  of  vaccination  they  become 
sufficiently  adapted  to  the  poison  to  be  able  to  manifest  their  normal 
inflammatory  reaction.  This  explanation  certainly 'harmonises  with 
the  fact  that  during  the  period  of  vaccinations  in  general  and  of 
vaccination  against  toxins  in  particular,  the  blood  usually  presents  a 
more  or  less  distinct  hyperleucocytosis.  Now,  as  is  well  known,  this 
1  "  Allgemeine  Therapie  der  Infectioriskrankheiten,"  Berlin  u.  Wien,  1899,  S.  1052. 


Artificial  immunity  against  toxins  353 

phenomenon    of    hyperleucocytosis    is    one   of    the   most   striking 
manifestations  of  a   positive  chemiotaxis  in  white  corpuscles.    It  is 
true  that,  as  to  this  reaction  during  the  course  of  vaccination,  the 
views  of  observers  are  not  unanimous.    Besredka1,  as  the  outcome  of 
his  work  on  this  subject,  expresses  himself  very  distinctly.     "During 
the  course  of  an  immunisation  against  diphtheria  toxin,"  he  writes, 
"one  always  observes  a  marked  reaction  in  the  goat,  either  at  the 
beginning  or  at  an  advanced  stage  of  the  period  of  injections  and 
especially  in  the  first  few  hours  after  injection  "  (p.  322).   Nicolas  and 
Courmont2  in  their  first  memoir  maintain  that  hyperleucocytosis 
"is    not   necessary   for   immunisation."     Nevertheless,   in  the  de- 
scription of  their  experiments,   which  were  performed  on  horses 
vaccinated  against  diphtheria,  it  is  clear  that  the  number  of  white 
corpuscles  is  often  markedly  increased.      Further,  in  several  cases 
they  describe  the  formation  of  tumours  at  the  point  of  inoculation,  [37 1] 
some  of  which  end  in  suppuration.    Under  these  conditions,  it  is  not 
possible  to  deny  a  vaccinal  reaction  on  the  part  of  the  leucocytes. 
Later,  Nicolas,  Courmont  and  Prat3  published  a  second  memoir  on 
the  same  subject,  in  which  they  seek  to  confirm  their  view  of  the 
uselessness  of  hyperleucocytosis  in  vaccination  against  the  poison  of 
diphtheria.    They  give  details  of  experiments  on  several  species  of 
animals  and  insist  specially  on  the  conditions  in  which  they  have  not 
observed  hyperleucocytosis.    "  The  doses  from  the  first  have  always 
been  extremely  weak  and  with  the  addition  of  Lugol's  solution  to 
attenuate  them  ;  only  very  gradually  have  we  reached  stronger  doses, 
as  that  is  one  of  the  indispensable  conditions  for  the  avoidance  of 
leucocytic  variations,  whilst  obtaining  a  good  and  rapid  immunisa- 
tion"   (p.    974).    These    special   precautions   to   avoid  hyperleuco- 
cytosis demonstrate  clearly  that  this  phenomenon  is  usually  pro- 
duced   during    the    course    of    vaccination.      It    is    quite    natural 
that  we  should,  by  proceeding  very  slowly  and  with  small  doses 
of  toxin,   succeed  in  diminishing  or  even  suppressing  the  afflux 
of  leucocytes ;   but  this  fact  cannot  in  any  way  minimise  the  im- 
portance of  the  leucocytic  reaction  in  vaccination.    In  these  particular 
cases,  this  reaction  may  take  place  without  the  number  of  leucocytes 
in  the  blood  being  noticeably  increased.    In  reading  the  details  of 
the  experiments  made  by  the  Lyons  observers,  it  will  be  seen  that, 

1  Ann.  de  Vlnst.  Pasteur,  Paris,  1898,  t.  xn,  p.  318. 

2  Arch,  de  med.  exper.  et  cFanat.  path.,  Paris,  1897,  t.  ix,  p.  770. 

3  Journ.  de  physiol.  et  de  path,  gen.,  Paris,  1900,  t.  u,  p.  973. 

B.  23 


354  Chapter  XII 

in  spite  of  all  their  precautions,  they  were  unable  to  prevent  the 
production  of  hyperleucocytosis.  In  all  their  cases,  where  they  took 
the  precaution  to  count  the  leucocytes  several  times  a  day,  there  was 
an  undoubted  increase  of  these  cells.  We  may  here  recall  Salo- 
monsen  and  Madsen's  account  of  the  immunisation  of  a  horse  against 
diphtheria  toxin,  in  which  they  point  out  the  frequency  of  tume- 
factions and  even  of  abscesses.  In  most  cases  the  pus  was  sterile, 
which  renders  it  probable  that  the  white  corpuscles  had  accumulated 
at  the  seat  of  inoculation  as  the  result  of  some  influence  exerted  by 
the  diphtheria  toxin. 

By  far  the  most  important  and  remarkable  change  met  with  in 
animals  vaccinated  against  toxins  and  venoms,  consists  in  the  appear- 
ance of  antitoxic  power  in  their  blood  and  fluids  in  general.  This 
[372]  fact  was,  as  already  mentioned,  first  demonstrated  by  von  Behring 
and  Kitasato1  in  the  blood  of  rabbits  immunised  against  tetanus. 
The  blood  itself,  or  the  blood  serum,  mixed  with  a  quantity  of  tetanus 
toxin  more  than  sufficient  to  cause  fatal  poisoning,  sets  up  no  disease 
when  injected  into  animals.  In  their  earliest  researches,  von  Behring 
and  Kitasato  kept  the  mixtures  in  contact  in  vitro  for  24  hours, 
before  injecting  them  into  test  animals.  Later,  they  found  that  this 
prolonged  contact  outside  the  body  was  unnecessary  and  that  they 
could  obtain  successful  results  by  injecting  the  serum  of  vaccinated 
animals  and  the  toxin  simultaneously,  even  at  different  points  of  the 
body.  This  discovery  was  immediately  afterwards  applied  by  its 
authors  to  diphtheria  and,  in  the  case  of  both  intoxications,  confirmed 
by  numerous  observers. 

For  some  time  we  were  satisfied  with  vaccinating  small  laboratory 
animals  and  establishing  the  antitoxic  power  of  their  blood  serum ; 
later,  the  vaccination  of  large  animals,  especially  horses,  was  com- 
menced with  the  object  of  obtaining  large  quantities  of  anti tetanus 
and  antidiphtheria  serum  for  medical  use.  During  the  course  of 
these  experiments  the  principal  characters  of  the  antitoxic  fluids 
were  established.  It  was  deemed  desirable  to  isolate  the  antitoxic 
substance  from  the  blood  serum  in  order  to  get  rid  of  every  un- 
necessary and  inactive  admixture,  so  that  the  antitoxin  might  be 
used  in  as  pure  a  form  as  possible.  This  idea  of  isolating  the  anti- 
toxic substance  had,  however,  soon  to  be  abandoned  as  impossible  of 
realisation.  Antitoxin  is  a  non-cry stallisable  substance,  of  unknown 
chemical  composition,  which  adheres  firmly  to  the  albuminoid 
1  Deutsche  med.  Wchwchr.,  Leipzig,  1890,  S.  1113. 


Artificial  immunity  against  toxins  355 

substances  of  the  serum.  It  is  usually  regarded  as  belonging  to 
the  same  albuminoid  group  of  substances,  though  it  is  not  possible 
to  prove  this  satisfactorily.  Von  Behring1,  however,  who  studied 
this  question  in  collaboration  with  Knorr,  denies  the  albuminoid 
nature  of  tetanus  antitoxin.  After  demonstrating  that  this  anti- 
toxin, when  the  antitetanus  serum  is  submitted  to  dialysis,  passes 
through  the  dialysing  membrane,  these  observers  found  that  they 
could  not  obtain  the  characteristic  reactions  of  albuminoids  in  the 
clialysed  fluid.  It  must  be  admitted,  however,  that  this  negative 
result  is  not  sufficient  to  justify  a  denial  of  the  albuminoid  nature  of  [373] 
antitoxin.  When  Nencki  and  Mme  Sieber2  sought  to  produce  the 
reactions  of  albuminoid  substances  with  the  digestive  juice  of 
Nepenthes  (the  well-known  insectivorous  plant)  they  got  no  result ; 
but  after  the  concentration  of  the  juice  in  vacua,  it  at  once  gave  the 
characteristic  reaction  with  nitric  acid,  and  also  with  acetic  acid, 
potassium  ferrocyanide  and  Millon's  reagent. 

The  antitoxins  may  be  precipitated  along  with  the  globulins  and 
are  distinguished,  in  general,  by  a  fairly  great  resistance  against 
physical  and  chemical  influences.  In  this  respect  they  are  allied  to  the 
agglutinins,  the  fixatives  and  the  precipitins,  considered  elsewhere,  and 
are  sharply  distinguished  from  the  cytases.  The  antitoxins  resist 
temperatures  which  destroy  the  cytases  and  remain  unaltered  to 
beyond  60° — 65°  C.  They  are  more  stable  than  the  delicate  toxins  of 
tetanus  and  diphtheria,  but  they  are  more  easily  altered  than  the 
toxins  of  cholera,  of  Bacillus  pyocyaneus  and  the  venoms.  When 
stored  in  a  dry  state  in  the  residue  of  evaporated  serums  and  protected 
from  light  and  air,  the  antitoxins  will  keep  for  a  very  long  time 
without  showing  any  notable  attenuation.  This  property  is  very 
important  in  practice. 

The  antitoxins,  in  this  respect  also  resembling  the  fixatives  and 
the  agglutinins,  are  humoral  substances  in  the  strictest  sense  of  the 
term.  They  are  found  not  only  in  prepared  serums  but  abound  also 
in  the  plasma  of  the  circulating  blood,  and  in  the  plasmas  of  the 
lymph  and  of  exudations.  Vaillard  and  Roux3  have  shown  that  the 
clear  acellular  serous  fluid  of  the  oedema  produced  by  the  slowing 
of  the  circulation  in  rabbits  vaccinated  against  tetanus  toxin,  is  as 
antitoxic  as  the  blood  itself.  Even  the  aqueous  humour  of  a  strongly 

1  "Die  praktischen  Ziele  der  Blutserumtherapie,"  Leipzig,  1892,  S.  52. 

2  Ztschr.f.physiol.  Chem.,  Strassburg,  1901,  Bd.  xxxn,  8.  318. 
8  Ann.  de  VInst.  Pasteur,  Paris,  1893,  t.  vn,  p.  81. 

23—2 


356  Chapter  XII 

immunised  animal  is  antitoxic,  though  to  a  less  degree.  On  the 
other  hand,  the  saliva  and  urine  exhibit  very  little  antitoxic  power, 
even  when  they  are  derived  from  animals  hyperimmunised  against 
tetanus  toxin.  Milk,  as  first  demonstrated  by  Ehrlich1,  is  fairly  rich 
in  antitoxin,  although  much  less  so  than  the  blood.  According  to 
the  estimation  of  Ehrlich  and  Wassermann2,  in  the  same  immunised 
[374]  animal,  milk  contains  one-fifteenth  to  one-thirtieth  of  the  amount  of 
diphtheria  or  tetanus  antitoxin  contained  in  the  blood.  Pus  is  always 
less  antitoxic  than  blood  or  blood  serum.  According  to  Roux  and 
Vaillard  (/.  c.,  p.  82),  the  pus  of  their  rabbits  vaccinated  against 
tetanus  toxin  was  only  one-sixth  or  one-eighth  as  antitoxic  as  the 
serum  of  the  blood.  In  Salomonsen  and  Madsen's3  antidiphtheritic 
horse  the  cellular  sediment  of  the  pus  was  about  one-half  as  antitoxic 
as  the  blood. 

For  the  development  of  the  antitoxic  property  in  the  fluids  of  the 
body,  it  is  not  essential  that  animals  should  belong  to  species  sus- 
ceptible to  the  corresponding  toxin.  Animals  naturally  most  refrac- 
tory against  the  poisons  of  diphtheria  and  tetanus  are  also  capable 
of  producing  antitoxins.  Vaillard4  demonstrated  this  fact  in  the 
fowl.  This  bird,  which  is  naturally  refractory  against  tetanus,  usually 
acquires  a  very  marked  antitetanic  power  in  its  blood  after  one 
or  more  injections  of  tetanus  toxin.  He  observed,  however,  that, 
in  fowls  thus  treated,  at  a  stage  when  the  fluids  of  the  body  are  anti- 
toxic, the  albumen  of  the  egg  is  not  so.  The  antitoxin,  therefore, 
does  not  pass  into  this  nutritive  secretion,  as  it  does  into  the  milk 
of  mammals.  On  the  other  hand,  as  has  been  demonstrated  by 
F.  Klemperer5,  the  vitellus  of  the  eggs  of  fowls  treated  with  tetanus 
toxin  in  time  acquires  an  antitoxic  property  of  the  most  marked 
character. 

The  antitoxins,  found  especially  in  the  fluids  of  the  body  but 
only  scantily  in  the  cells,  exert  some  action  on  the  toxins.  What 
is  the  nature  of  this  action?  This  question,  much  studied  and 
discussed,  is  one  of  very  great  importance  in  connection  with  the 
general  problem  of  acquired  immunity  against  toxins.  In  his  first 
memoir,  written  in  collaboration  with  Kitasato,  von  Behring  (Deutsche 

1  Ztschr.f.  Hyg.,  Leipzig,  1892,  Bd.  xii,  S.  183. 

2  Ztschr.f.  Hyg.,  Leipzig,  1894,  Bd.  xvm,  S.  248. 

1  Ann.  de  VInst.  Pasteur,  Paris,  1 897,  t.  xi,  p.  324. 

4  Compt.  rend.  Soc.  de  UoL,  Paris,  1891,  p.  462 ;  Ann.  de  VInst.  Pasteur,  Paris, 
1892,  t.  vi,  p.  229. 

5  Arch.f.  exper.  Path.  u.  Pharmakol,  Leipzig,  1893,  Bd.  xxxi,  S.  371. 


Artificial  immunity  against  toxins  357 

med.  WchnscJir.,  Leipzig,  1890,  S.  1113)  formulates  his  first  thesis  as 
follows  :  "  the  blood  of  a  rabbit  immunised  against  tetanus  possesses 
the  property  of  destroying  tetanus  toxin/'  This  idea  of  destruction, 
which  would  remove  all  toxic  power  from  the  poison,  would  naturally 
present  itself  to  the  mind  and  wras  at  once  accepted  by  a  great  many 
observers,  but  the  numerous  facts  now  accumulated  on  the  subject  [375] 
will  not  allow  us  to  accept  a  real  destruction  of  toxins  by  antitoxins. 
Tizzoni1  was  one  of  the  first  to  point  out  certain  contradictions 
between  the  theory  of  destruction  and  the  phenomena  produced  in 
animals  injected  with  tetanus  toxin  and  antitoxin.  Buchner2  also 
brought  forward  new  facts  wrhich  led  him  to  conclude  that  antitoxin, 
instead  of  acting  directly  on  the  toxin,  exerts  its  influence  exclu- 
sively on  the  living  elements,  thus  protecting  the  animal  against 
intoxication.  Amongst  the  arguments  advanced  by  the  Munich 
observer,  the  principal  one  is  drawn  from  the  different  action  of 
mixtures  of  tetanus  toxin  and  antitetanus  serum  on  various  species 
of  animals.  It  has  been  clearly  shown  that  the  guinea-pig  is  more 
susceptible  to  tetanus  than  is  the  mouse.  In  poisoning  with  tetanus 
toxin  it  requires  an  absolutely  larger  quantity  of  toxin  to  kill  the 
guinea-pig  than  to  kill  the  mouse.  But  if  we  take  into  account  the 
weight  of  these  animals,  the  conditions  change  completely.  Thus,  to 
cause  a  fatal  tetanus  in  a  guinea-pig,  which  weighs  twenty  times  more 
than  a  mouse,  we  need  only  inject  into  the  former  a  dose  at  most  ten 
times  greater  than  that  necessary  to  produce  fatal  intoxication  in  the 
mouse.  Buchner  prepared  a  mixture  of  tetanus  toxin  and  anti- 
tetanus  serum  which,  in  the  mouse,  produces  no  tetanic  phenomenon 
or  only  sets  up  feeble  and  transient  symptoms.  According  to  the 
theory  of  direct  action,  we  must  assume  that  in  this  mixture  the 
toxin  is  completely  or  almost  completely  neutralised  by  the  antitoxin 
of  the  serum.  But  when  Buchner  injected  the  same  quantity  of  mix- 
ture into  guinea-pigs,  without  increasing  it  in  proportion  to  the  greater 
weight  of  these  animals,  he  produced  a  tetanus  of  the  most  marked 
character.  There  has,  consequently,  remained  in  the  mixture  a 
sufficient  amount  of  free  toxin,  whose  tetanigenic  action  is  mani- 
fested in  the  guinea-pig,  an  animal,  as  we  have  seen,  more  susceptible 
than  the  mouse.  Buclmer's  experiment  has  been  verified  by  several 
observers.  Roux  and  Yaillard3  carried  out  others  which  afford 

1  JBerl.  klin.  Wchnschr.,  1893,  S.  1266. 

2  Miinchen.  med.  Wchnschr.,  1893,  S.  480. 

3  Ann.  de  VInst.  Pasteur,  Paris,  1894,  t  vin,  p.  725. 


358  Chapter  XII 

similar  evidence.  The  same  mixture  of  tetanus  toxin  and  specific 
serum  which  is  borne  without  the  least  difficulty  by  normal  guinea- 
pigs,  causes  typical  tetanus  in  other  guinea-pigs  of  the  same  weight, 
and  apparently  in  the  best  of  health,  but  which  have  been  im- 
[376]  munised  some  time  before  against  the  Massowah  vibrio.  In  another 
series  of  experiments,  Roux  and  Vaillard  injected  into  guinea- 
pigs  a  very  large  amount  of  antitetanus  serum  "capable  of  im- 
munising them  thousands  of  times/'  and,  shortly  afterwards,  a  lethal 
dose  of  tetanus  toxin.  The  normal  guinea-pigs  were  thoroughly 
resistant  to  this  test,  whilst  several  guinea-pigs  into  which  were  also 
injected  the  products  of  other  micro-organisms,  acquired  tetanus. 
Analogous  results  were  obtained  with  mixtures  of  diphtheria  toxin 
and  aritidiphtheria  serum.  Roux  concludes  from  these  facts  "that 
the  antitoxins  act  on  the  cells."  Against  the  theory  of  the  destruc- 
tion of  toxins  by  antitoxins,  he  invokes  the  influence  of  heat  on 
mixtures  of  these  two  substances.  Calmette1,  under  Roux's  inspi- 
ration and  in  his  laboratory,  carried  out  various  experiments  on  anti- 
venomous  serum.  A  mixture  of  this  with  snake  venom,  in  such 
proportion  that  the  poison  became  inactive,  regained  its  toxicity 
after  being  heated  for  five  minutes  at  68°  C.  A  normal  animal,  in- 
jected with  this  mixture,  succumbed  as  if  it  had  received  pure 
venom.  On  being  heated  at  68°  C.  the  serum  lost  all  its  antitoxic 
power  over  the  venom,  and  the  latter,  which  only  becomes  modified  at 
a  much  higher  temperature,  remained  intact.  Later,  a  similar  result 
was  obtained  by  Wassermann2  in  his  experiments  with  pyocyanic 
toxin.  This  poison  is  resistant  at  even  higher  temperatures  than  is 
snake  venom,  whilst  the  antitoxin  of  the  serum  is  destroyed  under 
the  same  conditions  as  are  the  other  antitoxins.  Taking  advantage 
of  these  peculiarities,  Wassermann  boiled  the  mixture  of  pyocyanic 
toxin  and  antitoxin  serum,  being  careful  to  dilute  it  with  two  volumes 
of  distilled  water  before  doing  so.  This  mixture  which,  before  it  was 
heated,  was  quite  innocuous  for  guinea-pigs,  again  became  a  fatal 
poison  after  the  destruction  of  the  antitoxin. 

These  experiments  prove  clearly  that,  in  the  action  of  the  anti- 
toxin on  the  toxin,  there  can  no  longer  be  any  question  of  an  actual 
destruction  of  the  latter,  a  view  which  has  been  accepted  by  both 
von  Behring  and  Ehrlich.  But,  as  pointed  out  by  Roux  at  the 

1  "Le  venin  des  serpents,"  Paris,  1896,  p.  58. 

2  Ztschr.f.  Hyg.,  Leipzig,  1896,  Bd.  xxn,  S.  263. 


Artificial  immunity  against  toxins  359 

International  Congress  at  Budapest  in  1894,  the  manifestation  of 
the  toxic  action  of  the  venom  after  it  has  been  heated  along  with 
antitoxin,  may  be  reconciled  with  the  view  that  the  combination 
between  the  two  substances,  if  such  take  place,  must  be  very  unstable. 
This  same  remark  may  be  applied  to  Wassermann's  experiment.  [377] 
Therefore  the  great  majority  of  observers,  if  not  all,  admit  that  the 
antitoxin  combines  with  the  toxin  to  form  an  innocuous  and  unstable 
substance  which  can  be  decomposed  by  heat  and  by  other  agents. 
The  researches  on  the  action  of  antitoxins  in  vitro  have  had  a 
powerful  influence  in  determining  this  view. 

We  have  already  in  Deiiys  and  van  de  VeldeV  experiments  an 
indication  of  the  direct  action  of  certain  antitoxins.  These  observers 
showed  that  the  serum  of  animals  vaccinated  against  a  Staphylococcus 
is  capable  of  neutralising  in  vitro  a  particular  toxin  to  which  van  de 
Velde  gave  the  name  of  leucocidin.  When  it  was  added  to  a  drop  of 
the  exudation  from  a  rabbit,  this  leucocidin  in  a  very  short  time 
destroyed  the  white  corpuscles,  by  dissolving  the  cell  content  but 
leaving  the  nucleus  untouched.  When  Denys  and  van  de  Velde 
prepared  mixtures  of  leucocytes,  leucocidin  and  antileucocidic  serum 
in  vitro,  the  white  corpuscles  retained  their  normal  condition  for  a 
very  long  time.  The  leucocidin  was,  therefore,  rendered  inactive  by 
the  direct  influence  of  the  corresponding  antitoxin.  These  facts  have 
been  confirmed  by  Bail2  and  other  observers  and  even  extended  to 
certain  other  microbial  toxins.  Thus,  the  Bacillus  pyocyaneus  pro- 
duces a  leucocidin  which  kills  the  white  corpuscles  and  dissolves 
their  contents3.  With  the  object  of  facilitating  experiments  with 
these  leucocytic  poisons  and  the  corresponding  antitoxic  serums, 
Neisser  and  Wechsberg4,  of  the  Institute  of  Experimental  Thera- 
peutics at  Frankfort,  invented  a  method  which  allows  us  to  observe 
the  phenomena  of  the  destruction  of  the  leucocytes  and  of  the  anti- 
toxic power  in  test  tubes,  without  having  recourse  to  a  microscopical 
examination.  They  applied  the  fact,  discovered  by  Ehrlich,  that 
living  formed  elements  reduce  methylene  blue  and,  depriving  it  of  its 
oxygen,  decolorise  it.  Leucocytes  from  aseptic  exudations  are  intro- 
duced into  tubes  and  a  weak  solution  (2%)  of  methylene  blue  is 
poured  on  them.  To  prevent  the  re-oxidation  of  this  colouring- 

1  La  Cellule,  Lierre  et  Lou  vain,  1896,  t.  xi,  p.  359;  Ann.  de  llnst.  Pasteur,  Paris, 
1896,  t.  x,  p.  580. 

2  Arch.f.  Hyg.,  Miinchen  u.  Leipzig,  1897,  Bd.  xxx,  S.  348. 

3  Gheorghiewsky,/4rzn.  de  Vlnst.  Pasteur,  Paris,  1899,  t.  xin,  p.  298. 

4  Ztschr.f.  Hyg.,  Leipzig,  1901,  Bd.  xxxvi,  S.  330. 


360  Chapter  XII 

matter  by  the  oxygen  of  the  air,  the  surface  of  the  fluid  is  covered 
[378]  with  a  layer  of  liquid  paraffin.  If  the  leucocytes  are  living,  the 
lower  blue  layer  becomes  decolorised  in  a  short  time  (in  about  two 
hours) ;  when  the  corpuscles  are  dead,  decoloration  does  not  take 
place.  By  adding  to  the  mixture  of  leucocytes  and  colouring  matter 
some  leucocidin,  alone  or  along  with  antileucocidic  serum,  it  is  possible 
not  only  to  observe  with  the  naked  eye  the  phenomena  which  take 
place  in  these  cases,  but  also  to  estimate  to  some  extent  the  pro- 
portions of  poison  and  counterpoison. 

All  these  researches  make  it  clear  that  the  antitoxin  acts  directly 
on  the  leucocidin.  Similar  facts  have  been  noted  as  regards  cer- 
tain other  organic  poisons  and  their  antitoxins.  Shortly  after  the 
discovery  of  antileucocidin  by  Denys  and  van  de  Velde,  Kanthack 
made  a  communication  to  the  Physiological  Society  in  18961,  ex- 
hibiting tubes  in  which  the  coagulating  action  of  Cobra  venom  on 
the  blood  had  been  prevented  by  the  addition  of  anti venomous 
serum.  Of  all  the  experiments,  however,  made  to  prove  the  direct 
action  of  antitoxin  on  toxin,  Ehrlich's2  have  played  the  most  important 
part  in  the  study  of  this  question.  Ehrlich  directed  his  attention  to 
ricin  which,  as  Robert  demonstrated,  has  the  property  of  agglutinat- 
ing the  red  corpuscles  of  defibrinated  blood.  This  phenomenon  can 
be  easily  observed  in  vitro.  In  tubes  containing  red  blood  corpuscles, 
the  addition  of  ricin  causes  these  corpuscles  to  agglutinate  into 
clumps  and  to  fall  to  the  bottom  of  the  tube,  leaving  a  clear  superna- 
tant fluid.  After  adding  progressively  increasing  quantities  of  antiricic 
serum  to  the  tubes  containing  fluid  blood  and  ricin,  Ehrlich  was  able 
to  demonstrate  that  small  quantities  of  antiricin  merely  retarded  the 
precipitation  of  the  red  corpuscles,  whilst  larger  doses  completely  pre- 
vented it.  Having  studied  the  proportions  of  ricin  and  its  antidote, 
necessary  to  retard  and  prevent  the  fatal  poisoning  of  animals,  Ehrlich 
was  struck  by  the  parallelism  which  is  exhibited  between  the  action 
of  the  antitoxin  in  the  living  animal  and  that  in  the  test  tubes. 

The  study  of  anticytotoxins,  discussed  in  the  fifth  chapter,  has 
furnished  another  opportunity  of  observing  the  action  of  antitoxins 
[379]  in  vitro.  Camus  and  Gley  and  H.  Kossel  were  the  first  to  observe 
the  action  in  vitro  of  antitoxic  serum  against  the  ichthyotoxin  of 
eel's  serum.  Since  this  observation,  this  phenomenon  has  been 
repeatedly  studied  in  the  antihaemolysins  and  antispermotoxins. 

1  [At  a  meeting  held  at  St  Bartholomew's  Hospital,  London,  cited  by  Stephens 
and  Myers  in  Journ.  Path,  and  Bacterial,  Edin.  and  London,  1898,  vol.  v,  p.  280.] 

2  Fortschr.  d.  Med.,  Berlin,  1897,  Jahrg.  xv,  S.  41. 


Artificial  immunity  against  toxins  361 

The  antidiastatic  serums  also  act  in  vitro  and,  as  their  effect  can  be 
demonstrated  on  soluble  ferments  placed  in  contact  with  unorganised 
bodies,  such  as  gelatine  and  casein,  the  purely  chemical  character  of 
the  reaction  is  all  the  more  strikingly  shown.  We  are  indebted  to 
von  Dungern,  Briot  and  Morgenroth  for  accurate  observations  on  this 
subject. 

Martin  and  Cherry1  made  use  of  a  different  method  to  demon- 
strate the  direct  action  of  antitoxins  on  toxins  which  exhibit  their 
toxic  power  on  the  animal  organism.  They  chose  snake  venom  mixed 
with  an ti venomous  serum.  The  mixtures  were  filtered  under  great 
pressure  [50  atmospheres]  through  a  film  of  gelatine,  under  the  idea 
that,  if  the  venom  and  antitoxin  were  not  chemically  combined,  the 
former  alone,  owing  to  its  much  smaller  molecules  as  compared  with 
those  of  the  anti venom,  would  pass  into  the  filtered  fluid.  This  fluid 
should,  under  these  conditions,  possess  a  toxic  power  for  animals, 
when  the  mixture,  used  for  filtration,  was  deprived  of  the  larger 
molecules.  Martin  and  Cherry  left  the  venom  and  the  antitoxic 
serum  in  contact  for  periods  of  varying  length,  before  filtering  the 
mixtures.  As  the  result  of  a  series  of  such  experiments  carried  out 
according  to  this  scheme,  they  found  that  the  product  of  the  filtra- 
tion made  after  some  minutes'  contact  between  the  two  substances, 
was  distinctly  toxic  ;  whilst  the  filtrate  obtained  after  a  contact  of 
half-an-hour  was  absolutely  innocuous.  From  their  observations 
these  authors  conclude  that  the  antitoxin  enters  into  chemical  com- 
bination with  the  venom,  but  that  the  combination  does  not  take 
place  instantaneously,  a  certain  amount  of  time  being  necessary  for 
its  accomplishment. 

In  addition  to  the  time  factor  others  have  an  influence  on  the 
combination  between  toxins  and  antitoxins,  as  is  seen  from  Ehrlich's2 
and  Knorr's3  investigations.  Both  observers  have  shown  that  anti- 
toxin neutralises  the  toxin  more  slowly  in  dilute  solutions  than  in 
more  concentrated  form.  For  this  reason,  when  animals  are  injected  [380] 
with  very  weak  solutions,  the  toxin  may  manifest  its  action  before  it 
can  be  neutralised  by  the  antitoxin ;  this  may  lead  to  erroneous 
conclusions.  On  the  other  hand,  according  to  data  furnished  by 
these  authors,  temperature  also  exerts  an  influence  on  the  combi- 

1  Proc.  Roy.  Soc.  London,  1898,  Vol.  LXIII,  p.  423. 

2  Klin.  Jahrbuch.,  Berlin,  1897,  Bd.  vi,  S.  13  [of  reprint]. 

3  Fortschr.  d.Med.,  Berlin,  1897,  Jahrg.  xv,  S.  657;  Miinchen.  med.  Wchnschr., 
1898,  S.  321. 


362  Chapter  XII 

nation.  Lowering  the  temperature  retards,  whilst  raising  it  accelerates 
the  neutralisation  of  the  toxins  by  the  antitoxins.  Insisting  on  the 
purely  chemical  character  of  the  combination  between  these  two 
substances,  Ehrlich  and  Knorr  adduce  the  fact  that  this  combination, 
in  cases  where  we  have  a  complete  neutralisation  of  the  toxin,  follows, 
most  rigorously,  the  law  of  multiple  doses,  that  is  to  say,  in  order  to 
render  innocuous  a  hundred  doses  of  toxin  we  have  only  to  take  a 
hundred  times  the  quantity  of  antitoxin. 

The  series  of  facts  summarised  above  demonstrate  distinctly  that 
antitoxins  act  directly  on  toxins.  But  how  can  this  result  be  recon- 
ciled with  the  observations  given  above  according  to  which  must  be 
admitted  the  no  less  real  influence  of  the  organism  of  the  living 
animal  on  intoxication  by  mixtures  of  antitoxin  with  toxin  ?  Knorr1 
sought  at  first  to  minimise  the  importance  of  the  facts  brought  for- 
ward by  Buchner  and  Roux.  He  failed  to  corroborate  Buchner's 
results  and  found  that  the  injection  of  mixtures,  made  with  very 
large  doses  of  tetanus  toxin  (20,000  times  the  minimal  lethal  dose) 
and  corresponding  quantities  of  antitetanus  serum,  brought  about 
the  same  effect  in  guinea-pigs  and  mice.  By  modifying  the  quantity 
of  antitoxin,  he  rendered  the  mixture  equally  innocuous  or  equally 
toxic  for  these  two  species.  But  the  data  given  by  Knorr  are  quite 
sufficient  to  prevent  us  from  accepting  his  conclusion.  In  his  experi- 
ments, as  in  those  of  Buchner,  the  guinea-pigs  manifested  a  greater 
susceptibility  and  died  from  mixtures  which,  in  mice,  caused  merely  a 
tetanus  of  medium  intensity. 

Some  have  sought  to  explain  Buchner's  experiment  by  assuming 
that  the  mixtures,  lethal  for  the  guinea-pig  and  innocuous  for  the 
mouse,  owed  their  toxic  action  to  the  presence  of  the  tetanus  toxone 
and  not  of  the  true  tetanus  poison,  the  tetanospasmin.  This  hypo- 
thesis of  toxones,  as  stated  above,  was  put  forward  by  Ehrlich  as  the 
[381]  outcome  of  his  ingenious  researches  on  the  constitution  of  the  diph- 
theria poison.  As,  however,  the  toxones  must  act  differently  from  the 
toxins,  we  can  only  attribute  to  their  action  the  results  in  those  cases 
where  the  guinea-pigs  die  without  presenting  typical  symptoms  of 
true  tetanus,  that  is  to  say  without  spasms.  Now,  in  Buchner's 
experiments,  a  much  larger  proportion  of  these  animals,  injected 
with  the  same  mixtures  as  the  mice,  succumbed  and  exhibited  the 
characteristic  tetanic  convulsions.  Even  in  those  cases,  however, 

1  "  Experimentelle  Untersuchungen  iiber  die  Grenzen  der  Heilungsmoglichkeit 
des  Tetanus,"  Marburg,  1895,  SS.  14,  21. 


Artificial  immunity  against  toxins  363 

where  the  death  of  the  guinea-pigs  might  be  attributable  to  an 
intoxication  by  the  toxone,  the  general  result  could  not  be  altered. 
The  toxones  are,  according  to  Ehrlich,  manufactured  by  the  micro- 
organisms in  the  culture  media  and  form  an  integral  part  of  the 
natural  microbial  poisons.  Again,  they  are  neutralised  by  antitoxic 
serums.  If,  therefore,  in  spite  of  there  being  the  same  quantity  of 
toxones  and  of  antitoxin  in  the  mixtures,  these  mixtures  become 
more  toxic  for  the  guinea-pig  than  for  the  mouse,  we  have  an  indica- 
tion that  some  special  change  must  take  place  in  the  animal  to  upset 
the  conditions  of  toxicity. 

Weigert1  accepts  the  accuracy  of  Buchner's  experiment,  which, 
indeed,  can  no  longer  be  denied,  but  explains  it  on  the  hypothesis 
that  there  is  some  substance  in  the  animal  possessing  a  very  great 
affinity  for  the  toxin.  This  substance  is  supposed  to  be  capable  of 
decomposing  the  innocuous  combination  of  the  antitoxin  with  the 
toxin,  just  as  heat  does  in  Calmette's  and  Wassermann's  experiments, 
described  above.  In  both  cases  the  toxin  would  be  set  free  to  exert 
its  noxious  action.  Such  a  hypothesis  is  very  probable,  because  it 
agrees  with  direct  observation,  but  it  compels  us  to  accept  some  new 
phenomenon  which  is  produced  not  in  vitro,  but  in  the  living  animal, 
and  which  carries  on  its  work  in  a  very  different  fashion  in  the 
guinea-pig  and  in  the  mouse. 

In  the  present  imperfect  state  of  our  knowledge  it  is  very  difficult 
to  form  any  idea  of  the  precise  conditions  which  must  intervene  in 
the  organism  of  the  guinea-pig  to  cause  the  tetanus  toxin  to  act  in  a 
mixture  with  antitoxin  which  is  much  more  innocuous  for  the  mouse. 
In  order,  however,  to  satisfy  those  who  seek  to  understand  these 
complex  phenomena,  it  may  be  useful  to  cite  another  example  of 
antitoxic  action  in  which  certain  factors  are  distinguished  by  their 
simplicity. 

Lang,  Heymans  and  Masoin2  have  demonstrated  that  hyposul-  [382] 
phite  of  soda  prevents  poisoning  by  prussic  acid.  This  terrible 
poison  becomes  innocuous  if  we  take  care  to  introduce  into  the 
animal  by  any  channel  whatever  (subcutaneously,  intravenously,  or  by 
the  stomach)  a  sufficient  quantity  of  hyposulphite  of  soda.  Under 
these  conditions  the  sulphite  is  substituted  for  the  hydrogen  of  the 
prussic  acid,  transforming  the  poison  into  sulphocyauic  acid,  which 

1  Lubarsch  u.  Ostertag's  "  Ergebuisse  d.  allgera.  Pathologie  u.  patholog.  Aiiato- 
mie,"  Wiesbaden,  iv  Jahrg.  (for  1897),  S.  121. 

2  Arch,  inlernat.  de  Pharmacodyn.,  Gaud  et  Paris,  1896,  Vol.  m,  p.  77. 


364  Chapter  XII 

has  no  action  on  the  organism.  The  hyposulphite  of  soda,  then,  acts 
as  the  antitoxin  of  the  prussic  acid,  thanks  to  a  chemical  reaction  of 
substitution  between  bodies  of  simple  composition.  We  have  never 
yet  succeeded  in  reproducing  this  reaction  in  vitro,  whilst  in  the 
animal  body  it  is  effected  with  very  great  ease.  Consequently,  we  are 
quite  justified  in  invoking  special  conditions  in  the  body  of  the 
living  animal ;  this,  however,  does  not  preclude  the  possibility  of  a 
transformation  of  the  toxic  substance  into  an  innocuous  substance 
through  a  chemical  reaction.  It  is  probable  that  analogous  phe- 
nomena may  also  be  met  with  in  the  action  of  true  antitoxins  on 
the  microbial  toxins  or  allied  substances  (venoms,  vegetable  toxal- 
bumins). 

The  case  of  the  destruction  of  micro-organisms,  which  is  now  more 
easily  studied  because  it  is  possible  to  observe  with  the  eye  the  fate 
of  these  organisms  in  the  animal,  is  a  further  source  of  valuable 
information.  The  direct  action  of  cytases  on  certain  bacteria,  such  as 
the  cholera  vibrio,  can  be  just  as  easily  demonstrated  in  vitro  as  can 
the  action  of  antiricin  on  ricin.  If  we  proceeded  to  argue  from  this, 
a  perfectly  accurate  observation,  that  the  living  animal  plays  no  part 
in  the  destruction  of  the  micro-organisms  and  that  this  destruction 
takes  place  always  in  a  fashion  analogous  to  Pfeiffer's  phenomenon 
in  vitro,  we  should  undoubtedly  arrive  at  an  erroneous  conclusion. 
We  know  already,  as  has  been  indicated  in  previous  chapters,  that 
the  granular  transformation  of  vibrios  is  only  part  of  a  whole 
series  of  phenomena  of  destruction  of  micro-organisms,  the  great 
majority  of  which  phenomena  require  more  or  less  active  inter- 
vention of  the  animal  organism.  In  reality,  matters  usually  go  on  in 
a  very  complicated  fashion,  in  which  direct  and  indirect  actions  are 
blended  in  varied  proportions.  In  the  examples  described  elsewhere, 
we  see,  alongside  the  granular  transformation,  an  agglutination  into 
[383]  clumps  and  immobilisation,  and  an  ingestion  and  intracellular  de- 
struction of  micro-organisms.  The  final  phase,  no  doubt,  is  always 
a  chemical  or  physico-chemical  action,  exerted  against  the  micro- 
organism, but  how  varied  are  the  means  used  to  bring  about  this 
result !  We  may  surely  be  allowed  to  suppose  that- analogous  pheno- 
mena may  take  place  in  the  action  of  antitoxins  on  the  toxins. 

Just  as,  in  the  analysis  of  the  influence  of  serums  on  the  micro- 
organisms, it  was  found  useful  to  study  the  action  of  certain  fluids 
less  complicated  than  the  anti-infective  specific  serums,  so  we  may 
utilise  information  furnished  by  the  antitoxic  action  of  fluids  other 


Artificial  immunity  against  toxins  365 

than  the  true  antitoxins.  Cases  are  by  no  means  rare  in  which  nor- 
mal serums  exert  a  certain  influence  on  toxins.  Thus,  Pfeiffer1  noted 
that  the  normal  blood  serum  of  the  goat  has  the  power  to  prevent 
fatal  poisoning  by  the  cholera  toxin.  Freund,  Grosz  and  Jelinek2 
observed  an  analogous  action  of  solutions  of  nucleohiston  on  diph- 
theria intoxication  and  Kondratieff3  demonstrated  the  same  action  of 
an  extract  of  the  spleen  on  the  tetanus  poison.  Calmette4,  in 
collaboration  with  Delearde,  studied  the  influence  of  a  whole  series 
of  fluids  on  abrin  intoxication.  Whilst  physiological  saline  solution 
was  absolutely  incapable  of  preventing  the  death  of  animals,  fresh 
broth  exerted  an  undoubted  antitoxic  power.  Amongst  normal 
serums,  ox  serum  exhibited  a  certain  antirabic  property.  More, 
however,  than  the  serums  of  normal  animals,  have  those  of  animals 
immunised  against  various  toxins  other  than  abrin  (antitetanus,  anti- 
diphtheria,  anti venomous  serums,  &c.)  been  found  to  possess  the 
power  of  preventing  intoxication  by  abrin.  These  facts  are  connected 
with  others  of  analogous  nature,  previously  demonstrated  by  Calmette5, 
of  which  I  may  cite  the  following  :  the  serum  of  animals  vaccinated 
against  tetanus  toxin  is  active,  though  to  a  less  degree,  against  snake 
venom ;  the  serum  of  rabbits  vaccinated  against  rabies,  a  serum 
powerless  to  protect  against  this  disease,  is,  however,  very  markedly 
effective  against  the  same  venom ;  the  serum  of  animals  immunised 
against  snake  venom  is  also  antitoxic  against  scorpion  venom  (I  have  [334] 
myself  had  the  opportunity  of  confirming  this  fact  on  several 
occasions).  In  all  these  examples,  the  serums  have  proved  to  be  less 
efficacious  against  poisons  other  than  the  toxin  with  which  the 
animals  that  furnished  the  blood  had  been  treated.  Ehrlich6,  too, 
has  demonstrated  that  animals  vaccinated  against  robin  (toxalbumin 
of  Robinia  pseudacacia)  produce  a  serum,  antitoxic  not  only  against 
this  poison  but  also  against  ricin.  It  need  scarcely  be  added  that  in 
all  these  cases  of  non-specific  action  of  serums  derived  from  vaccin- 
ated animals,  no  question  of  any  antitoxic  effect  of  normal  serums 
can  enter.  In  all  the  experiments  just  summarised,  the  serums  of 
normal  animals,  used  as  controls,  were  found  to  be  inefficacious. 

1  Ztschr.f.  Hyg.,  Leipzig,  1895,  Bd.  xx,  S.  210. 

2  Centralblf.  inn.  Med.,  Leipzig,  1895,  Jahrg.  xvi,  SS.  913,  937. 

3  Arch.f.  exper.  Path.  u.  Pharmakol,  Leipzig,  1896,  Bd.  xxxvii,  S.  191. 

4  Ann.  de  VInst.  Pasteur,  Paris,  1896,  t.  x,  p.  703. 

5  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  225. 

6  "Die  Werthbemessung  d.  Diphtherieheilserums "  (Klin.  Jahrb.,  Berlin,  1897, 
p.  20  of  reprint). 


366  Chapter  XII 

If,  in  the  case  of  the  non-specific  action  of  serums,  it  were 
allowable  to  advance  the  hypothesis  of  a  direct  influence  of  these 
fluids  on  the  toxins,  it  would  still  be  impossible  to  sustain  this  view 
where  broth  fulfils  the  antitoxic  rdle.  This  fluid,  much  simpler  in 
composition  than  any  serum,  is  an  excellent  culture  medium  for 
micro-organisms  and  one  in  which  the  toxins  develop  well  and  can  be 
kept  for  a  fairly  long  period.  There  is,  therefore,  not  the  slightest 
ground  for  assigning  to  it  any  direct  antitoxic  action,  on  the  contrary, 
everything  leads  us  to  regard  it  as  an  indirect  agent,  which  acts  by 
stimulating  the  reaction  of  the  animal  organism.  Here,  then,  the 
case  would  be  quite  analogous  to  that  of  the  action  of  broth  as  a 
protective  agent  against  certain  bacterial  injections,  a  subject  already 
discussed  in  the  tenth  chapter.  In  this  same  category  of  indirect 
influences  also,  must  be  ranked  the  example  of  the  antitoxic  action 
of  the  blood  of  the  crayfish  against  scorpion  venom.  I  have 
demonstrated  in  a  series  of  experiments  that  the  fresh  blood  of  the 
crayfish  has  the  power  to  prevent  fatal  intoxication  of  mice  by 
scorpion  venom.  Injected  in  a  dose  of  from  1  to  1*25  c.c.,  several 
minutes  or  an  hour  before  the  injection  of  the  rapidly  fatal  dose  of 
scorpion  venom,  the  crayfish's  blood  exerts  a  very  distinct  preventive 
action.  It  might  be  supposed  from  this  that  the  crayfish  belongs  to 
the  group  of  animals  insusceptible  to  scorpion  venom.  This,  how- 
ever, is  not  the  case.  The  crayfish  is  very  susceptible  to  this  poison 
[385]  and  succumbs  to  a  quarter  the  dose  necessary  to  kill  a  mouse.  The 
blood  of  the  crayfish  is,  therefore,  completely  ineffective  as  a  pro- 
tective to  the  crayfish  itself,  and  only  exerts  its  action  when  introduced 
into  the  body  of  the  mouse.  It  might  be  concluded  that  it  is  only 
after  it  has  been  drawn  from  the  crayfish  that  the  blood  acquires  its 
antitoxic  power.  Experiment  contradicts  this  supposition.  Crayfish 
blood,  when  injected  into  another  crayfish,  in  equal  or  greater  amount 
than  is  necessary  to  protect  a  mouse,  is  incapable  of  preventing  fatal 
intoxication  by  scorpion  venom,  although,  here  again,  the  crayfish 
received  only  one-quarter  of  the  dose  of  venom  used  for  the  mice. 

We  are,  therefore,  compelled  to  believe  that  the  crayfish's  blood 
is  antitoxic  for  the  mouse,  not  in  virtue  of  its  djrect  neutralising 
action  on  the  venom,  but  owing  to  some  indirect  influence  on  the 
organism  of  the  mouse.  It  is  impossible  to  define,  exactly,  the 
mechanism  of  this  action.  We  may  suppose  that  the  blood  of  the 
crayfish  contains  some  substance  which,  by  itself,  is  insufficient  to 
prevent  the  intoxication,  but  which  becomes  active  in  the  presence  of 


Artificial  immunity  against  toxins  367 

some  other  substance,  also  inefficacious  by  itself,  met  with  in  the 
organism  of  the  mouse.  Here  we  should  have  something  analogous 
to  what  is  met  with  in  immunity  against  micro-organisms  where  both 
fixatives  and  cytases  intervene  to  bring  about  the  destruction  of 
micro-organisms.  By  making  researches  in  vitro  on  the  action  of 
the  fluids  on  bacteria,  we  may  easily  observe  certain  phenomena 
which  appear  to  indicate  their  direct  influence.  Take  the  case  of 
the  fluid  of  an  oedema  from  an  animal  vaccinated  against  the  cholera 
vibrio  which  renders  this  micro-organism  motionless  and  agglutinates 
it  in  vitro  ;  the  oedema  of  an  unvaccinated  animal  produces  no 
such  effect.  If,  however,  we  were  to  conclude  from  this  fact  that,  in 
the  oedema  of  the  living  animal  or  in  its  subcutaneous  tissue,  every- 
thing goes  on  as  in  the  test  tube  and  that  no  other  phenomenon  of 
reaction  against  the  vibrios  is  produced,  we  should  fall  into  a  grave 
error.  It  is  extremely  probable  that,  in  the  resistance  of  the  living 
animal  against  the  toxins,  the  phenomena  are  more  complicated  than 
are  those  observed  in  vitro.  The  example  of  the  blood  of  the  cray- 
fish which  prevents  the  poisoning  of  the  mouse,  without  having  any 
influence  on  that  of  the  crayfish  itself,  may  here  serve  as  a  guide  to 
us.  It  is  possible  that,  as  in  the  struggle  against  the  micro-organisms, 
we  have  here  a  co-operation  of  two  substances,  each  one  of  which, 
by  itself,  is  inactive.  One  of  these  substances  would  be  found  pre-  [386] 
existent  in  the  blood  of  the  crayfish,  the  other  forming  part  of  the 
organism  of  the  mouse.  Perhaps  the  action  of  this  blood  is  even 
more  complicated  and  only  becomes  active  through  the  mediation  of 
some  constituent  of  the  living  cell. 

Our  study  of  immunity  against  toxins  long  ago  revealed  cases  in 
which  this  resistance  cannot  be  attributed  simply  to  the  antitoxic 
action  of  the  body  fluids.  Animals  vaccinated  against  living  micro- 
organisms may  succumb  to  infection  in  spite  of  the  presence  of  a 
strong  anti-infective  power  of  the  body  fluids ;  similarly  animals 
immunised  against  toxins  may  die  from  intoxication  in  spite  of  the 
antitoxins  contained  in  their  fluids.  Facts  of  this  order  are  not  rare. 
Roux  and  Yaillard1  on  several  occasions  observed  animals  which  died 
from  tetanus  although  they  had  a  large  supply  of  antitoxin  in  their 
blood.  Von  Behriug2  and  his  collaborators,  Knorr,  Ransom,  and 

1  Ann.  de  VInst.  Pasteur,  Paris,  1893,  t.  vn,  p.  98. 

2  Deutsche  med,  Wchnschr.,  Leipzig,  1893,  S.  1253;  "Allgemeine  Therapie  der 
Infectionskrankheiten"  in  Eulenburg  u.  Samuel's  "Lehrb.  d.  allg.  Therapie,"  Berlin 
u.  Wien,  1899,  Bd.  in,  S.  1051. 


368  Chapter  XII 

Kitashima,  also  collected  a  large  number  of  analogous  facts.  They 
showed  that  horses  that  have  been  treated  for  a  long  time  with 
tetanus  toxin  and  whose  blood  serum  is  very  antitoxic,  still  experience 
marked  illness  after  fresh  injections  of  toxin  and  may  even  succumb, 
in  spite  of  the  presence  of  a  large  amount  of  antitoxin  in  their  blood. 
In  these  cases  the  morbid  phenomena  are  undoubtedly  different  from 
those  typical  of  tetanus.  Instead  of  the  muscular  contractions  which 
characterise  this  disease,  the  above  observers  noted  disturbance  in 
the  regulation  of  the  body  temperature,  exudative  inflammation 
around  the  point  of  inoculation,  impairment  of  appetite  and  fall  of 
body  weight.  Sometimes  they  observed  muscular  tremors  and  marked 
feebleness  in  the  movements.  These  symptoms  differing  from  those 
of  typical  tetanus,  it  may  be  asked  whether  this  poisoning  is  not  due 
to  special  substances  other  than  tetanus  toxin  in  the  fluids  injected. 
Von  Behring  does  not  think  that  this  is  the  case,  for  he  found  that 
by  adding  antitetanus  serum  the  formation  of  exudations  at  the  seat 
of  inoculation  was  suppressed.  These  exudations,  then,  must  be 
attributed  to  the  tetanus  toxin. 

In  the  cases  where  animals  immunised  against  diphtheria  toxin 
fall  ill  and  even  die  as  the  result  of  fresh  injections  of  toxin,  in  spite 
[387]  of  the  presence  of  a  large  quantity  of  antitoxin  in  their  blood,  we 
might  also  cast  doubts  on  the  diphtheritic  character  of  the  poisoning, 
because  the  clinical  picture  of  this  poisoning  is  not  a  very  typical  one. 
At  the  Pasteur  Institute,  where  a  large  supply  of  antidiphtheria 
serum  is  prepared,  we  see,  from  time  to  time,  horses,  which  have  long 
been  undergoing  the  process  of  immunisation  and  are  furnishing  a 
very  good  serum,  suddenly  fall  ill  and  die  from  intoxication,  without 
presenting  any  symptom  of  infective  disease.  On  one  occasion,  there 
was  actually  quite  a  small  epidemic  of  fatal  poisonings  as  the  result 
of  the  injection  of  a  quantity  of  diphtheria  toxin  not  exceeding  the 
doses  which  had  been  well  borne  previously.  Amongst  the  horses, 
inoculated  with  the  same  toxin,  five  of  the  best  furnishers  of  serum 
died.  The  others,  some  of  which  were  producing  only  a  weak  serum, 
remained  unaffected. 

Von  Behring  and  Kitashima1  have  given  a  detailed  history  of  a 
young  horse  which  had  become  very  susceptible  as  the  result  of 
vaccination  with  diphtheria  toxin.  It  finally  succumbed  to  the  intoxi- 
cation in  spite  of  the  presence  of  diphtheria  antitoxin  in  its  blood. 

If,  in  these  examples,  we  have  any  reason  to  doubt  the  specific 
1  Berl.  klin.  Wchnsclir.,  1901,  S.  157. 


Artificial  immunity  against  toxins  369 

nature  of  the  intoxication,  all  doubt  must  give  way  before  the  case 
described  by  Brieger1.  One  of  his  goats,  well-immunised  with  tetanus 
toxin,  which,  for  months,  had  furnished  a  good  serum  and  even  an 
anti tetanus  milk,  after  an  injection,  stronger  than  the  preceding  ones, 
was  seized  with  tetanic  contractions.  These,  becoming  general, 
brought  about  the  death  of  the  animal  with  the  symptoms  of  classic 
tetanus.  The  blood,  drawn  off  after  death,  exhibited  strong  antitoxic 
power. 

As  the  result  of  these  observations  von  Behring  formulated  the 
theory  of  a  hypersusceptibility  acquired  during  immunisation.  "  Para- 
doxical as  it  may  appear,"  he  writes2,  "there  can  no  longer  exist 
any  doubt  that  horses  which  have  acquired  a  high  immunity  as  the 
result  of  treatment  with  tetanus  toxin,  present  a  histogenic  hyper- 
susceptibility  of  the  organs  which  react  against  the  tetanus  toxin." 
In  support  of  this  thesis  von  Behring  compares  the  effect  produced 
by  this  toxin  on  horses  immunised  with  this  same  poison  and  on 
normal  horses  treated  with  antitoxic  serum  from  other  horses.  The 
former,  in  spite  of  the  fact  that  they  contain  in  their  blood  1,500  times  [388] 
more  antitoxin  than  do  the  latter,  are,  nevertheless,  less  refractory 
to  tetanus  toxin.  This  feeble  resistance  is  due,  in  von  Behring's 
opinion,  to  the  much  greater  susceptibility  of  the  living  elements  in 
the  horses  treated  with  repeated  doses  of  the  poison. 

Von  Behring's  theory  of  this  form  of  acquired  specific  hyper- 
susceptibility  has  been  confirmed  by  several  well-observed  facts. 
These  show  that,  in  the  animal  subjected  to  treatment  by  toxins, 
phenomena  of  very  diverse  order  are  evolved  simultaneously  :  on  the 
one  hand,  cell  reactions  which  bring  about  the  production  of  anti- 
toxins ;  on  the  other,  an  increase  in  the  susceptibility  of  some  of  the 
living  elements  to  the  specific  poison.  We  are,  however,  justified 
in  asking  if  the  great  difference  between  the  immunity  of  animals 
treated  with  toxin,  and  that  of  others  treated  with  antitoxic  serum, 
can  be  altogether  attributed  to  this  hypersusceptibility? 

Let  us  examine  in  a  little  more  detail  some  examples  of  this 
hypersusceptibility.  We  know  that  the  guinea-pig  is  characterised 
by  its  great  natural  susceptibility  to  the  toxins  of  tetanus  and  diph- 
theria. Small  doses  of  these  poisons  are  quite  sufficient  to  produce 
in  it  a  fatal  intoxication.  But  it  is  possible  to  diminish  greatly  this 

1  Ztschr.f.  Hyg.,  Leipzig,  1895,  Bd.  xix,  S.  109. 

2  "Allgemeine  Therapie  der  Infectionskrankheiten,"  in  Eulenburg  u.  Samuel's 
"Lehrb.  d.  allg.  Therapie,"  Berlin  u.  Wien,  1899,  Bd.  in. 

B.  24 


370  Chapter  XII 

feeble  resistance  of  the  guinea-pig  by  frequent  injections  of  very 
small  quantities  of  toxin.  Knorr1  increased  their  susceptibility  to 
tetanus  toxin  by  daily  injections  of  one-tenth  of  a  minimal  lethal 
dose.  The  animals  died  before  they  had  received  the  ten  tenths  of 
this  dose.  The  hypersusceptibility  produced  under  these  conditions 
might  be  so  great  that  one-fiftieth  of  the  minimal  lethal  dose  was 
capable  of  causing  death.  From  these  facts  we  can  understand 
the  great  difficulty  experienced  in  the  earlier  attempts  to  vaccinate 
guinea-pigs  by  means  of  unmodified  toxin. 

Yon  Behring  and  Kitashima2  made  analogous  researches  on  the 
susceptibility  of  guinea-pigs  to  diphtheria  toxin.  By  frequent  in- 
jections of  very  small  doses  of  this  poison  they  succeeded  in  killing 
these  animals  with  -^  of  the  minimal  lethal  dose  distributed  over 
several  injections.  They  never  succeeded  in  vaccinating  guinea-pigs 
with  increasing  doses  of  pure  diphtheria  toxin.  Their  animals  died 
even  when  they  commenced  with  one-millionth  of  the  minimal  lethal 
dose. 

[389]  Here,  then,  we  have  examples  of  the  greatest  hypersusceptibility 
that  it  is  possible  to  observe.  When  we  compare  it  with  the 
changes  in  the  antitoxic  power  of  the  blood,  we  find  that  these  are 
even  more  marked.  Thus,  Salomonsen  and  Madsen's  horse,  to  which 
we  have  already  referred,  presented  extraordinary  oscillations  in  this 
power.  After  receiving,  during  the  course  of  immunisation,  a  fresh 
dose  of  diphtheria  toxin,  the  antitoxic  value  of  its  blood  suddenly  fell 
more  than  one-third  (35  %)•  In  order  to  neutralise,  completely,  this 
dose  of  toxin,  when  injected  into  a  normal  animal  mixed  with  anti- 
toxic serum  from  this  same  horse,  a  very  small  quantity  of  the  blood 
of  the  latter  would  have  been  sufficient.  The  injection  into  the 
immunised  horse  should  have  passed  unperceived,  as  this  animal 
contained  in  its  body  more  than  50  litres  of  strongly  antitoxic  blood. 
Nevertheless  the  antitoxic  power  of  this  blood  fell  12,000  times 
more  than  it  ought  to  have  fallen  according  to  the  calculation  made 
upon  the  data  just  indicated.  This  fall  is  incomparably  greater  than 
the  increase  of  susceptibility  to  toxin  in  the  most  significant  examples 
reproduced  above. 

As  the  fact  above  cited  is  not  at  all  unique,  it  is  probable  that 
the  phenomena  which  appear  in  the  animal  subjected  to  vaccination 

1  "  Experimentelle  Untersuchungen  iiber  die  Grenzen  der  Heilungsmoglichkeit 
des  Tetanus,"  Marburg,  1895,  SS.  18,  19. 

2  Berl  klin.  Wchnschr.,  1901,  S.  157. 


Artificial  immunity  against  toxins  371 

by  toxins,  must  be  much  more  complicated  than  is  usually  sup- 
posed. If  the  fresh  injections  of  these  poisons  bring  about  a 
specific  hypersensitiveness  on  the  one  hand,  and  on  the  other  a  great 
fall  in  antitoxic  power,  followed  by  its  still  more  notable  augmentation, 
it  is  evident  that  the  introduction  of  toxins  must  give  rise  to  a  great 
perturbation  in  the  cell  functions.  The  general  analogy  between 
acquired  immunity  against  micro-organisms  and  against  toxins  pro- 
bably rests  on  similar  bases.  Kretz1  has  already  advanced  the 
hypothesis  that,  in  antitoxic  action,  two  factors,  comparable  to  the 
cytases  and  fixatives  in  the  antimicrobial  action,  co-operate.  In  the 
absence  of  one  of  these  elements  we  can  understand  that  the  one 
which  remains  may  be  incapable  of  bringing  about  the  neutralisation 
of  the  toxin.  For  this  reason  the  antitoxic  serum  may  act  very 
differently  in  the  organism  of  the  animal  which  produces  it  and  in 
that  of  a  normal  animal  which  receives  it.  An  explanation  which  is 
adequate  for  the  antitoxic  action  of  the  blood  of  the  crayfish  injected 
into  mice  serves  equally  well  in  the  case  of  the  antitoxic  influence  [390] 
of  the  serums  of  animals  which  themselves  succumb  to  intoxication. 

Wassermaim's2  experiments  on  the  anticytase  serums  might 
appear  to  supply  an  argument  against  the  hypothesis  we  are  de- 
fending. Having  shown  that  animals  injected  with  antityphoid  serum 
die  of  intoxication  when  serum  which  prevents  the  action  of  the 
cytases  is  introduced  simultaneously,  Wassennann  put  the  question : 
May  not  the  action  of  the  antitoxins  be  prevented  by  this  same  anti- 
cytase serum?  To  solve  this  point  he  injected  into  guinea-pigs  a 
mixture  of  antidiphtheria  serum  with  toxin  in  excess  and  a  fairly 
strong  dose  (3  c.c.)  of  anticytase  serum,  upon  which  we  have  already 
spoken  (see  Chapter  VII).  The  animals,  so  treated,  behaved  exactly 
as  did  the  animals  used  for  control  which  received  the  same 
quantities  of  antitoxin  and  toxin  but  without  the  addition  of  anti- 
cytase serum.  Wassermann  concludes  from  these  experiments  that 
the  exclusion  of  the  cytase,  contrary  to  what  takes  place  with  anti- 
microbial serums,  in  no  way  impedes  the  action  of  the  antitoxins. 
This  conclusion,  which  appears  at  first  sight  to  be  justified,  cannot, 
however,  be  accepted,  as  the  two  examples  chosen  by  Wassermann, 
typhoid  infection  and  diphtheria  intoxication,  differ  very  profoundly 
from  each  other.  In  the  former,  we  have  an  experimental  typhoid 
peritonitis  which  kills  the  control  animals  in  less  than  24  hours, 

1  Ztschr.f.  Heilh,  Berlin,  1901,  Bd.  xxn,  S.  1. 

2  Ztschr.f .  Hyg.,  Leipzig,  1901,  Bd.  xxxvii,  S.  194. 

24—2 


372  Chapter  XII 

whilst  the  second  is  diphtheria  in  which  the  controls  do  not  succumb 
until  the  sixth  day  after  injection.  The  effect  of  the  anticytase 
serum  being  only  very  transitory,  it  is  quite  natural  that  this  should 
manifest  itself  in  an  infection  of  short  duration  and  should  not  do  so 
in  a  slow  intoxication.  Besides,  Wassermann  himself  has  shown  that 
in  several  other  cases  of  immunity  against  micro-organisms  (the  bacilli 
of  influenza  and  of  leprosy)  the  injection  of  his  anticytase  serums  does 
not  interfere  with  the  perfect  resistance  of  the  animals.  But  even 
were  it  demonstrated  that  the  cytases  really  play  no  part  in  immunity 
against  toxins,  the  intervention  of  some  other  similar  factor  could 
always  be  evoked. 

The  analogy  between  immunity  against  micro-organisms  and  that 
against  toxins  may  facilitate  the  study  of  the  relations  between  the 
latter  and  the  antitoxic  power  of  the  body  fluids.  In  the  preceding 
[391]  chapters  we  have  described  examples  in  which  animals  possess  a 
protective  power  in  their  blood  but  are  not  refractory  to  the  corre- 
sponding infection ;  on  the  other  hand,  we  have  cited  cases  in  which 
acquired  antimicrobial  immunity  exists  without  the  blood  presenting 
any  appreciable  protective  power.  The  idea  of  measuring  acquired 
immunity  against  micro-organisms  by  the  measurement  of  the  pro- 
tective or  agglutinative  power  of  the  blood  must  therefore  be 
abandoned,  and  it  is  impossible  to  regard  immunity  against  toxins 
as  a  function  of  the  antitoxic  property  of  the  body  fluids.  As  we 
have  seen,  animals  completely  refractory  to  tetanus,  such  as  the 
cayman,  whose  immunity  does  not  depend  on  the  antitetanic  power  of 
the  blood,  develop  antitoxin  after  the  injection  of  toxin.  A  similar 
state  of  affairs,  but  less  pronounced,  has  been  demonstrated  by 
Vaillard  as  occurring  in  the  fowl.  The  fowl,  in  spite  of  its  very 
marked  natural  immunity  against  tetanus,  produces  antitetanin  as  the 
result  of  the  introduction  into  its  body  of  tetanus  toxin  ;  the  rabbit, 
on  the  other  hand,  a  susceptible  animal,  may  acquire  a  real  immunity 
without  the  development  of  any  antitoxic  power  in  its  fluids.  An 
additional  fact  was  noted  by  Vaillard1.  He  showed  that  the  repeated 
inoculation  of  tetanus  spores  along  with  a  small  quantity  of  lactic 
acid,  made  below  the  skin  of  the  tail  of  rabbits  procured  for  them  an 
immunity  against  tetanus  toxin,  although  no  antitoxic  property  ap- 
peared in  their  blood.  In  his  experiments,  one  hundred  volumes  of 
blood  serum  were  found  to  be  incapable  of  neutralising  a  single 
minimal  lethal  dose  of  the  toxin.  The  rabbit,  however,  still  remains 
1  Compt.  rend.  Soc.  de  biol.,  Paris,  1891,  p.  464. 


Artificial  immunity  against  toxins  373 

quite  capable  of  developing  antitetanic  power  in  its  fluids.  All  that 
is  necessary  is  to  inject  into  it  some  tetanus  toxin  heated  to  60°  C.  or 
treated  with  Lugol's  iodo-ioduretted  solution.  As  the  outcome  of  his 
researches  Vaillard  concludes  that  the  antitoxic  property  of  the  body 
fluids  "is  not  sufficient... for  the  general  interpretation  of  acquired 
immunity,  as  it  cannot  be  demonstrated  in  all  animals  which  have 
become  refractory." 

The  facts  I  have  just  mentioned  were  demonstrated  early  in  our 
study  of  the  antitoxic  power  of  the  animal  organism.  Since  then  a 
large  number  of  analogous  data  have  been  collected.  Recently,  von 
Behring  and  Kitashima1  have  had  to  abandon  the  immunisation  of 
monkeys  against  diphtheria  toxin  because  of  the  poor  yield  in  anti-  [392] 
toxin  which  they  obtained.  The  blood  of  one  of  their  monkeys  that 
had  acquired  a  resisting  power  against  very  large  doses  of  diphtheria 
toxin  showed  only  a  very  moderate  antitoxic  power.  In  establish- 
ments where  antitoxic  serums  are  prepared  on  a  large  scale  the 
workers  have  become  convinced  that  the  yield  of  antitoxin  has  no 
direct  constant  ratio  to  the  immunity  of  the  animal.  This  has  been 
demonstrated  repeatedly  at  the  stables  of  the  Pasteur  Institute. 
Thus,  of  two  horses,  treated  at  the  same  time  and  in  exactly  the 
same  way  with  diphtheria  toxin,  one  furnished  a  very  good  antitoxic 
serum  which  was  maintained  at  200  units  Ehrlich,  rising  up  to  400 
units,  whilst  the  other  never  reached  150  units2.  And  yet  both  these 
animals  possessed  the  same  immunity  against  diphtheria  toxin.  They 
tolerate  considerable  doses  of  toxin  and  react  merely  by  a  slight  or 
insignificant  rise  in  temperature.  In  another  series  of  horses,  which 
have  been  immunised  for  nearly  seven  years,  one  remained  capable  of 
yielding  a  large  quantity  of  antitoxin,  seeing  that  the  value  of  its 

1  Berlin  klin.  Wchnschr.,  1901,  S.  157.   The  idea  of  immunising  monkeys  against 
diphtheria  was  suggested  to  von  Behring  by  the  fact  that  the  immunity  conferred  by 
serums  was  the  more  durable  the  nearer  the  relation  between  the  serum  used  and 
the  blood  of  the  species  which  receives  the  protective  injection.    Von  Behring 
supposed  that  the  diphtheria  antitoxin,  introduced  into  the  human  body,  would  be 
maintained  there  longer,  if  the  antitoxic  serum  injected  came  from  monkeys,  species 
much  nearer  man  than  is  the  horse,  the  usual  source  of  antidiphtheria  serum.     The 
immunity  conferred  by  this  horse  serum  is  generally  of  very  short  duration. 

2  Ehrlich's  antitoxic  unit  is  adopted  by  most  investigators  not  only  in  Germany, 
but  also  in  other  countries.     This  unit  corresponds  to  1  c.c.  of  serum  capable  of 
neutralising  100  lethal  doses  of  a  standard  toxin,  i.e.  that  used  to  establish  the  first 
standard  of  antitoxin.    The  serum  must  be  injected  after  being  mixed  in  vitro  with 
the  toxin.     The  neutralisation  must  be  complete  and  give  rise  to  no  symptom  of 
intoxication. 


374  Chapter  XII 

serum  oscillated  between  200  and  300  units.  After  five  years  of  this 
state  of  things  the  antitoxic  power  began  to  fall  considerably,  with- 
out, however,  any  corresponding  loss  of  immunity.  Indeed,  an 
injection  of  250  c.c.  of  toxin  (of  which  0'002  c.c.  was  sufficient  to  kill 
a  guinea-pig)  began,  at  the  commencement  of  the  present  year,  to  be 
borne  without  the  least  febrile  reaction.  An  attempt  was  made  to 
raise  the  antitoxic  power  of  the  blood  by  making  intravenous  in- 
jections of  toxin  and  of  diphtheria  culture,  but  in  vain.  The  yield  of 
antitoxin  continued  to  fall  and  it  became  necessary  to  employ  this 
horse  for  another  purpose  than  the  preparation  of  antidiphtheria 
serum.  This  is  by  no  means  an  isolated  example.  Of  a  large  number 
[393]  of  treated  horses  it  frequently  happens  that  certain  individuals,  with- 
out being  particularly  susceptible  to  a  given  toxin,  are  found  to  be 
incapable  of  producing  any  corresponding  antitoxin1. 

In  presence  of  the  fact  that  animals  very  resistant  to  toxins  may 
possess  no,  or  only  an  insignificant  antitoxic  power  in  their  fluids, 
and  that,  on  the  other  hand,  animals  in  which  this  property  is 
highly  developed  may  succumb  to  intoxication,  it  may  be  readily 
understood  that  immunity  against  toxins  and  the  antitoxic  power 
of  the  body  fluids  may  be  two  distinct  conditions.  Von  Behring 
has  clearly  demonstrated  the  fact  of  the  cellular  hypersensitive- 
ness  of  the  animal  immunised  against  the  corresponding  toxin 
and  has  laid  great  stress  upon  this  fact.  He  came2  to  the  con- 
clusion that  "the  immunity  of  the  tissues  and  the  production  of 
antitoxin  follow  a  parallel  course  in  their  development  so  slightly 
that,  in  spite  of  an  abundant  accumulation  of  antitoxin,  the  suscepti- 
bility of  the  elements  of  the  tissues  may  increase  in  an  extraordinary 
fashion."  If,  during  the  course  of  immunisation,  this  susceptibility 
can  increase  so  greatly,  it  is  probable  a  priori  that  under  certain 
circumstances  it  might  also  diminish  notably.  After  demonstrating 
"that  in  time  the  antitoxin  disappears  from  the  blood  of  animals 
immunised  with  toxins  without  any  consequent  disappearance  of 
immunity,"  von  Behring  formulated  the  conclusion  that  in  these 
animals  "the  living  elements  of  the  animal,  which  were  previously 
susceptible  to  the  poisons,  have  acquired  an  insusceptibility  towards 
the  same  substances."  This  result  fully  accords  with  the  facts  of  the 
change  of  the  negative  chemiotaxis  of  phagocytes  into  positive 

1  These  observations  were  communicated  to  me  by  M.  Prevot,  the  director  of  the 
serotherapeutic  station  of  the  Pasteur  Institute  at  Garches. 

2  Deutsche  med.  Wchnschr.,  Leipzig,  1893,  SS.  1253,  1254. 


Artificial  immunity  against  toxins  375 

chemiotaxis  for  micro-organisms  during  the  acquisition  of  anti-in- 
fective immunity. 

Later,  von  Behring1  changed  his  opinion.  Whilst  still  accepting 
the  change  of  cellular  susceptibility  in  the  direction  of  hypersensi- 
tiveuess  in  animals  immunised  against  toxins,  he  refused  to  admit 
the  change  in  the  opposite  direction.  The  cells,  according  to  him, 
never  lose  any  of  their  susceptibility,  so  that  acquired  immunity 
against  toxins  cannot  be  obtained  otherwise  than  by  means  of  [394] 
antitoxins  capable  of  neutralising  the  poison  in  a  susceptible  or 
hypersusceptible  animal.  This  new  theory  von  Behring  upheld 
in  several  papers  and  it  is  met  with  in  his  most  recent  publications. 
Nevertheless,  certain  well-established  facts  compel  us  to  accept  an 
immunity  against  toxins  as  coming  about  as  the  result  of  a  diminu- 
tion of  the  susceptibility  of  the  vaccinated  animal.  Parallel  with  his 
researches  on  the  increase  of  the  susceptibility  of  guinea-pigs  to 
tetanus  toxin,  researches  discussed  above,  Knorr2  describes  analogous 
experiments  on  rabbits.  When  these  animals  are  injected  with 
fractious  of  the  minimal  lethal  dose,  frequently  repeated,  the 
rabbit  not  only  does  not  become  hypersusceptible  to  tetanus  but 
exhibits  a  greater  and  greater  insusceptibility.  Whilst  guinea- 
pigs,  treated  according  to  this  method,  die  from  tetanus  before 
they  have  reached  the  minimal  lethal  dose,  rabbits,  as  the  result  of 
frequent  injections  of  small  quantities  of  tetanus  toxin,  become 
capable  of  resisting  five  times  the  lethal  dose  (for  normal  rabbits) 
without  exhibiting  the  slightest  symptom  of  illness.  Against  the 
attribution  of  this  result  to  the  acquired  insusceptibility  of  the  living 
animals  it  might  be  objected  that  the  immunity,  in  this  case,  may 
depend  on  the  antitoxic  power  of  the  fluids  of  the  body,  developed 
with  great  rapidity.  Such  an  objection  cannot  be  raised  in  the  case 
of  horses  which  become  insusceptible  to  toxins  after  a  long  period 
of  vaccination.  The  horse  whose  history  was  given  above,  when 
discussing  the  diminution  of  antitoxic  power,  may  serve  as  an  ex- 
ample. At  the  commencement  of  its  vacciual  period,  in  1894,  it 
reacted  to  the  injection  of  10  c.c.  of  diphtheria  toxin  by  a  rise  of 
temperature  of  1°  C.  Four  years  later,  when  its  blood  had  become 
very  antitoxic  (350  units  per  c.c.),  it  was  necessary  to  inject  350  c.c. 

1  Article  "Immunitat"  in  Eulenburg's  Realencydopadie,  III**  Aufl.,  Wien,  1896 ; 
see   also  his  "  Allgemeine  Therapie  d.   Infectionskrankheiten,"  in   Eulenburg  u- 
Samuel's  "Lehrb.  d.  allg.  Therapie,"  Berlin  u.  Wien,  1899,  Bd.  in,  SS.  996,  997. 

2  Op.  cit.  supra  p.  370,  S.  19. 


376  Chapter  XII 

of  toxin  to  obtain  the  same  rise  of  temperature.  Quite  recently, 
having  now  lost  the  greater  part  of  its  humoral  antitoxic  power, 
this  horse  exhibited  no  rise  of  temperature  after  an  injection  of 
250  c.c.  of  strong  diphtheria  toxin.  The  diminution  of  the  specific 
susceptibility  is  produced  in  this  case  in  a  most  marked  fashion ;  it 
is  not  therefore  to  the  antitoxic  property  of  the  body  fluids  that 
this  case  of  immunity  must  be  attributed. 

The  insusceptibility  acquired  against  poisons  of  different  kinds 
is  observed  also  in  cases  where  the  adaptation  is  not  accompanied 
[395]  by  the  production  of  humoral  antitoxic  properties,  as  in  the  im- 
munity of  frogs  against  abrin.  This  form  of  immunity  may  be  traced 
through  the  organic  series  down  to  such  lowly  developed  organisms 
as  the  plasmodium  of  the  Myxomycetes,  which  as  we  have  seen 
readily  becomes  adapted  to  different  poisons  (see  Chapter  II). 

It  can  be  clearly  seen,  then,  that  immunity  against  toxic  sub- 
stances is  a  very  complex  phenomenon  which  it  is  impossible  to 
reduce  simply  to  an  antitoxic  function  of  the  fluids  of  the  body. 
For  this  reason  we  cannot  accept  a  theory  which  would  confine  this 
kind  of  immunity  within  the  narrow  limits  of  a  simple  reaction 
between  two  substances,  a  reaction  quite  comparable  to  that  observed 
in  a  test-tube.  Attempts  have  been  made  to  determine  with  almost 
mathematical  precision  the  conditions  under  which  it  is  possible  to 
communicate  to  the  animal  a  resistance  against  microbial  toxins  and 
formulae  have  been  constructed  to  define  these  conditions.  But 
the  application  of  these  formulae  has  been  found  to  be  a  much  more 
difficult  matter.  In  Prussia,  with  the  sanction  of  the  Government, 
regulations  have  been  enacted  as  to  the  procedure  to  be  followed 
in  the  testing  of  antitoxic  serums,  and  a  paragraph  has  been  added 
which  requires  a  post-mortem  examination  of  the  guinea-pigs  em- 
ployed for  this  purpose  in  the  case  of  diphtheria  antitoxin.  "The 
dead  animals,"  says  this  instruction,  "must  be  submitted  to  a  post- 
mortem examination,  and  special  attention  must  be  directed  to  the 
presence  of  any  pre-existing  diseases  (tuberculosis,  pseudotuber- 
culosis,  pneumonia)  which  may  have  induced  hypersusceptibility  in 
the  animals  under  experiment."  Do  we  not  see  in,  this  a  proof  of 
the  important  intervention  of  the  organism  of  the  living  animal 
which  may  modify  the  results  of  calculations  based  upon  too  rigorous 
formulae?  It  must  not  be  forgotten,  too,  that  in  addition  to  the 
three  diseases  named  in  the  instructions,  we  have  a  number  of  other 
factors  which  may  influence  the  receptivity  and  the  resistance  of 


Artificial  immunity  against  toxins  377 

animals.  We  have  already  cited  Roux  and  Vaillard's  experiments 
which  demonstrated  that  even  animals  which  have  been  previously 
subjected  to  vaccinal  inoculations  against  certain  micro-organisms, 
exhibit  a  hypersusceptibility  to  mixtures  of  toxins  with  antitoxins. 

In  view,  then,  of  this  complexity  of  the  phenomena  of  acquired  im- 
munity against  toxins,  it  would  be  very  important  could  we  learn 
something  of  the  nature  and  origin  of  antitoxins.  Unfortunately, 
as  we  shall  see,  these  questions  are,  as  yet,  far  from  having  received 
a  satisfactory  solution. 

Struck  by  the  fact  that  antitoxins  exert  a  specific  action  on  the 
toxin  which  has  been  employed  in  the  treatment  of  the  animals  that 
produce  the  serum,  certain  observers  have  sought  an  explanation  on  [396] 
the  hypothesis  of  a  transformation  of  toxin  into  antitoxin.  We  have 
already  seen  that  antitoxic  action  is  not  always  absolutely  specific ; 
we  have  serums  which  prevent  intoxication  by  various  kinds  of 
poisons,  e.g.  antitetanus  serum,  which  is  active  against  both  tetanus 
toxin  and  snake  venom.  There  is,  however,  a  great  quantitative 
difference  between  the  influence  of  the  antitoxin  on  the  toxin  with 
which  the  animals  have  been  prepared  and  on  a  different  poison. 
Thus,  in  the  example  just  cited,  in  order  to  neutralise  snake  venom 
it  is  necessary  to  use  a  much  larger  quantity  of  antitetanus  serum 
than  against  the  toxin  of  tetanus.  The  classical  example  of  the 
specific  influence  of  antitoxins  is  the  absolute  inactivity  of  anti- 
diphtheria  serum  against  tetanus  and  the  same  non-effect  of  anti- 
tetanus  serum  against  diphtheria  intoxication.  The  most  simple 
explanation  of  this  specificity  of  action  appeared  to  be  the  sup- 
position that  each  antitoxin  contains  a  part  of  the  corresponding 
toxin,  modified  by  the  organism  of  the  animal.  H.  Buchner1  advo- 
cates this  hypothesis.  I  myself2  said  "that  it  is  probable  that 
antitoxins,  at  least  in  great  part,  represent  a  modification  of  the 
toxins  prepared  by  certain  cells  in  the  animal  body;  this  product 
is  then  poured  into  the  blood."  This  view  was  stated  as  a  "  pro- 
bability "  and  consequently  contains  no  affirmation  in  the  least 
definitive.  I  was,  therefore,  quite  prepared  to  give  it  up  under 
the  weight  of  the  crushing  criticism  formulated  by  several  very 
distinguished  observers.  It  was  objected ;  first,  that  antitoxin  is 
produced  by  animals  in  very  great  disproportion  to  the  quantity  of 
toxin  they  have  received ;  secondly,  that  the  animals  which  receive 

1  Milnchen.  med.  Wchnschr.,  1893,  S.  380. 

2  "  Immunitat "  in  Weyl's  "  Haiidbuch  der  Hygiene,"  Jena,  1897,  Bd.  ix,  S.  48. 


378  Chapter  XII 

an  injection  of  antitoxin  eliminate  it  from  their  body  much  more 
rapidly  than  do  those  which  prepare  it  in  their  own  body;  thirdly, 
that  antitoxins  are  sometimes  found  in  the  blood  of  healthy  animals, 
who  have  had  no  attack  of  the  disease  nor  any  injection  of  the 
specific  toxin.  Let  us  examine  these  objections  more  closely,  ob- 
jections all  based  on  well-established  facts. 

It  has  been  shown  that  the  antitoxin  produced  by  the  animal  is 
sufficient  to  neutralise  a  quantity  of  toxin  much  greater  than  that 
which  was  injected  into  the  animals  supplying  the  antitoxic  serum. 
[397]  Knorr1,  from  his  experiments,  calculated  that  a  horse  reacts  to  one 
unit  of  toxin  by  the  production  of  100,000  units  of  antitoxin.  This 
statement  certainly  does  not  allow  us  to  affirm  that  all  the  antitoxin 
corresponds  to  toxin,  but  it  does  not  eliminate  the  possibility  that 
toxin,  subjected  to  the  influence  of  the  cells  of  the  animal  body, 
may  be  found,  in  a  modified  form,  in  the  product  of  these  elements. 
This  hypothesis  would  be  quite  sufficient  to  explain  the  very  remark- 
able specificity  of  antitoxins. 

If  the  toxin,  in  order  to  be  modified  by  the  living  cells,  must  be 
subjected  to  some  special  action  on  the  part  of  the  latter,  we  can 
readily  understand  that  this  process  must  demand  a  greater  or  less 
length  of  time;  this  would  lead  to  a  much  slower  elimination  of 
the  antitoxin  than  in  the  case  where  it  had  been  injected,  ready 
prepared,  into  a  normal  animal.  From  the  commencement  of  his 
researches  on  immunity  against  poisons,  Ehrlich2  distinguishes  two 
kinds  of  this  immunity,  an  active  immunity  which  is  obtained  as 
the  result  of  the  introduction  of  toxins  into  the  animal,  and  a 
passive  immunity,  another  form  of  the  refractory  condition  which  is 
set  up  by  the  injection  of  antitoxic  serum  formed  in  the  actively 
immunised  animal.  Von  Behring3  applies  the  term  isopathic  im- 
munity to  active  immunity,  and  to  passive  immunity  that  of  antitoxic 
immunity.  It  is  generally  admitted  that  the  first  kind  of  immunity 
is  more  slowly  acquired,  but  that  it  persists  for  a  much  longer  period 
than  the  second  (passive  or  antitoxic  immunity)  which  is  acquired 
immediately  after  the  introduction  of  the  antitoxin,  but  which,  on 
the  other  hand,  lasts  for  a  short  time  only.  This  view  is  supported 
by  numerous  observations  on  the  very  rapid  disappearance  of  the 

1  Miinchen.  med.  Wchnschr.,  1898,  p.  321. 

2  Deutsche  med.   Wchnschr.,  Leipzig,  1891,  SS.   976,  1218;  [Ztschr.  f.  Hyg., 
Leipzig,  1892,  Bd.  xu,  S.  183]. 

3  "Allgemeine  Therapie  der  Infectionskrankheiten "  in  Eulenburg  u.  Samuel's 
"  Lehrbuch  der  allgemeine  Therapie,"  Berlin  u.  Wieii,  1899,  Bd.  in,  S.  997. 


Artificial  immunity  against  toxins  379 

refractory  condition.  According  to  von  Behriug  the  great  difference 
in  the  duration  of  the  isopathic  and  antitoxic  immunities  is  only  an 
apparent  one.  It  is  due  to  the  fact  that  antitoxins  are  usually 
introduced  along  with  the  serum  of  different  species  which  sets  up 
a  strong  reaction  and  is  rapidly  eliminated  from  the  animal.  Thus 
the  antitoxic  serum  of  the  horse  is  usually  injected  into  small 
animals  such  as  guinea-pigs,  rabbits,  and  mice.  When,  however, 
von  Behring  injected  horses  with  antitoxic  serums  from  other  horses,  [398] 
the  antitoxic  immunity  lasted  almost  as  long  as  in  animals  vaccinated 
with  toxins.  Ransom1  has  developed  this  thesis  in  a  work  carried  out 
in  von  Behring's  Institute  at  Marburg,  and  supports  it  by  compara- 
tive researches  which  demonstrate  the  more  rapid  disappearance  of 
the  antitoxin  when  introduced  with  the  serum  of  a  different  species 
than  when  introduced  with  that  of  the  same  species. 

Even  should  we  accept  the  current  view  on  the  greater  duration 
of  the  antitoxic  power  of  the  blood  in  isopathic  immunity,  the  hypo- 
thesis of  the  transformation  of  toxin  by  the  cells  of  the  animal  is 
not  necessarily  invalidated.  If  a  part  of  the  toxin  introduced  into 
the  animal  remains  stored  for  some  time  in  an  organ  it  is  evident 
that  only  gradually  can  it  be  subjected  to  the  transforming  action 
of  the  cells.  It  is  impossible,  in  the  present  state  of  our  knowledge, 
to  demonstrate  this  proposition,  but  we  may  invoke  in  its  favour  the 
prolonged  persistence  of  red  blood  corpuscles  when  introduced  into 
the  body  of  a  different  species  of  animal  (see  Chapter  IV).  These 
corpuscles  are  in  the  end  always  completely  digested  but  the  process 
is  of  long  duration. 

The  same  hypothesis  will  also  explain  a  fact,  first  demonstrated 
by  Roux  and  Yaillard2.  They  have  shown  that  after  repeated  bleed- 
ings of  rabbits  immunised  against  tetanus,  the  antitoxic  property 
of  the  blood  was  soon  raised  to  almost  the  same  value  as  before. 
Salomonsen  and  Madsen3  have  confirmed  the  fact  of  the  regene- 
ration of  antitoxin  after  the  bleeding  of  their  animals  (horses  and 
goats)  immunised  against  diphtheria.  Those  authors  who  do  not 
accept  the  possibility  of  the  transformation  of  toxins  in  the  production 
of  antitoxins,  regard  these  facts  as  absolutely  incompatible  with  the 
hypothesis  which  they  attack.  Thus,  Weigert4  considers  that  the 

1  Journ.  Path,  and  Bacterial,  Edin.  and  London,  1900,  Vol.  VI,  p.  180. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1893,  t.  vn,  p.  82. 

3  Ann.  de  VInst.  Pasteur,  Paris,  1898,  t.  xn,  p.  763. 

4  Op.  cit.  supra,  p.  363,  iv  Jahrg.,  S.  122. 


380  Chapter  XII 

regeneration  of  antitoxin  after  bleeding  can  only  be  understood 
by  accepting  that  antitoxin,  like  the  blood,  may  be  reproduced  in 
the  actively  immunised  animal  without  any  fresh  introduction  of 
toxin.  It  is,  however,  just  as  simple,  we  think,  to  explain  the  fact 
in  question  by  the  hypothesis  of  a  provision  of  toxin  stored  up  in 
[399]  certain  cells.  This  also  is  sufficient  explanation  of  another  observa- 
tion made  by  Salomonsen  and  Madsen1,  who  showed  that  pilocarpin 
is  capable  of  augmenting  the  production  of  antitoxin.  Since  it  is 
the  living  cells  which  transform  the  toxin  and  excrete  the  antitoxin, 
it  is  quite  natural  to  suppose  that  every  factor  which  stimulates  cell 
function  may  be  capable  of  causing  an  increase  of  the  product  trans- 
formed by  the  cells. 

The  third  argument  invoked  against  the  possibility  of  the  trans- 
formation of  toxins  into  antitoxins  is  based  on  the  fact  that  the 
serum  of  normal  horses  has  sometimes  a  certain  degree  of  antitoxic 
power  against  diphtheria  toxin.  The  horses  have  never  suffered 
from  diphtheria,  therefore  the  antidiphtherin  of  their  blood  has 
nothing  to  do  with  diphtheria  toxin.  It  is  not  known  why  the 
blood  serum  of  certain  untreated  horses  is  from  the  first  active 
against  diphtheria  toxin,  whilst  that  of  others  exerts  absolutely  no 
action  on  the  same  poison.  We  know  only  that  this  property  is  far 
from  being  constant  in  the  equine  species.  Perhaps  it  is  acquired 
as  the  result  of  the  penetration  into  the  animal  of  some  pseudo- 
diphtheria  bacillus,  whose  frequency  and  number  are  very  great. 
In  order  that  the  microbial  products  may  give  rise  to  the  formation 
of  antibodies,  it  is  not  at  all  necessary  that  the  micro-organisms 
should  produce  an  evident  disease.  Thus,  to  cite  one  example  only, 
Foerster2  observed  a  considerable  agglutinative  power  against  the 
typhoid  cocco-bacillus  in  the  serum  of  a  child  which  was  found 
living  among  a  family  of  typhoid  patients  but  which,  itself,  pre- 
sented no  morbid  symptom. 

The  criticism,  directed  against  the  hypothesis  that  modified  toxin 
enters  into  the  production  of  antitoxin,  may  not  be  sufficient  to  show 
the  incorrectness  of  this  view ;  it  does  not  follow,  however,  that  the 
view  is  right.  In  the  present  state  of  our  knowledge  it  is  impossible  to 
solve  the  problem  definitely,  and  as  the  hypothesis  of  transformation 
gives  us  the  best  idea  of  the  specificity  of  the  action  of  antitoxins, 
it  has  a  right  to  be  taken  into  consideration  as  much  as  any  other. 

1  Compt.  rend.  Acad.  d.  sc.,  Paris,  1898,  t.  cxxvi,  p.  1229. 

2  Ztschr.f.  Hyg.,  Leipzig,  1897,  Bd.  xxiv,  S.  514, 


Artificial  immunity  against  toxins  381 

Ehrlich1  has  formulated  another  hypothesis  to  explain  not  only 
this  specificity  but  the  origin  of  antitoxins  in  general.  This  is  the  [400] 
ingenious  hypothesis  of  side-chains  or  of  receptors,  which  has  already 
been  considered  in  other  chapters  of  this  work.  It  is  now  for  the 
first  time  brought  forward  in  relation  to  the  antitoxins  properly 
so-called,  that  is  to  say  substances  capable  of  preventing  intoxication 
by  microbial  toxins.  In  order  to  make  his  hypothesis  as  clear  as 
possible  Ehrlich  begins  by  explaining  its  bearing  on  the  concrete 
example  of  tetanus  antitoxin.  "  When  we  introduce  into  an  animal  a 
small  quantity  of  tetanus  toxin,  it  is  easy  to  obtain  exact  proof  that 
it  is  quickly  fixed  by  the  central  nervous  system,  probably  by  the 
motor  cells  of  the  ganglia ;  that  the  central  nervous  system  more 
than  any  other  organ  attracts  the  tetanus  toxin  and  retains  its  toxic 
molecules  very  firmly."  There  we  have  the  side-chains  of  the  proto- 
plasm fulfilling  this  role  and  subjecting  the  living  protoplasm  to  the 
prolonged  action  of  the  poison.  Once  it  is  combined,  the  side-chain 
becomes  incapable  of  fulfilling  its  normal  function,  and  there  is 
induced  on  the  part  of  the  living  elements  the  production  of  new 
chains  of  a  similar  character.  Following  the  law  that  the  reaction 
is  stronger  than  the  action,  there  is  an  over-production  of  these 
side-chains  which  finally  so  embarrass  the  cell  which  has  developed 
them  that  they  are  excreted  by  it  into  the  blood  plasma.  Once 
expelled  into  this  plasma,  they  continue  to  manifest  their  affinity 
for  the  tetanus  toxin,  an  aifinity  which  must  be  even  greater  in 
the  case  where  the  chains  are  found  in  the  blood  than  when  they 
were  connected  with  the  cell.  Owing  to  this  affinity,  these  chains, 
now  in  the  blood,  fix  the  tetanus  poison  introduced  into  the  animal 
and  prevent  it  from  reaching  the  susceptible  nerve  elements.  Anti- 
toxins, according  to  this  hypothesis  are,  therefore,  nothing  but  over- 
plus side-chains  poured  into  the  body  fluids.  Ehrlich  extends  his 
theory  to  a  whole  series  of  bodies  capable  of  causing  the  formation 
of  antitoxins  and  antidiastases.  "It  is  probable/'  he  says,  "that  all 
analogous  bodies  can  only  become  toxic  to  the  animal  on  condition 
that  the  animal  is  capable  of  fixing  their  toxophore  groups  in  certain 
of  the  organs  that  are  important  for  its  life  "  (p.  17). 

According  to  this  theory  tetanus  antitoxin  must  pre-exist  in  the 
central  nervous  system  of  the  normal  animal.  In  the  immunised 
animal,  the  side-chains  must  be  reproduced  in  very  great  quantity 

1  "Die  Werthbernessung  des  Diphtherieheilsemms "  (Klin.  Jahrb.,  Berlin,  1897, 
Bd.  vi),  SS.  13—17  of  reprint. 


382  Chapter  XII 

[401]  in  the  nerve  cells  and  pass  thence  into  the  circulation.  Indeed, 
Wassermann,  a  supporter  of  this  theory,  made  a  search  for  tetanus 
antitoxin  in  the  nerve  centres  of  normal  animals.  In  collaboration 
with  Takaki1  he  made  the  important  discovery  that  the  brain 
and  spinal  cord  of  small  mammals  (guinea-pigs  and  rabbits)  when 
triturated  with  tetanus  toxin  prevent  the  manifestation  of  its  toxic 
action  in  animals  most  susceptible  to  tetanus.  The  brain  was  always 
found  to  be  more  active  than  the  spinal  cord.  The  property  of 
neutralising  the  toxin  of  tetanus  belongs  to  the  solid  parts  of  the 
nerve  centres ;  the  fluid  of  the  cerebral  emulsion  is  incapable  of 
exercising  this  action. 

This  discovery  was  soon  confirmed.  Ransom2  demonstrated  it 
almost  at  the  same  time,  and  independently  of  Wassermann  and 
Takaki ;  and  the  fact  is  indisputable.  It  remains  to  be  seen  whether 
the  "  antitoxin  "  of  the  nerve  centres  of  normal  animals  is  really  the 
same  as  that  which  is  found  in  the  fluids  of  animals  immunised  against 
tetanus  toxin,  as  is  accepted  by  Wassermann  and  the  other  partisans 
of  the  side-chain  theory.  The  former  is  characterised  by  a  very  local 
reaction ;  it  is  incapable  of  being  dissolved  and  distributed  through 
the  body  of  the  animal.  This  is  shown  by  Marie's3  experiments,  and 
my  own4,  all  carried  out  in  my  laboratory.  All  that  is  necessary  is  to 
introduce,  beneath  the  dorsal  surface  of  the  thigh  of  a  guinea-pig,  a 
quantity  of  the  cerebral  substance  sufficient  to  neutralise  several 
times  the  lethal  dose  of  toxin,  and  below  the  skin  of  the  ventral 
aspect  of  the  same  thigh,  a  lethal  dose  of  this  toxin,  when  it  will  be 
found  that  the  guinea-pig  contracts  a  fatal  tetanus.  The  antitoxic 
action  of  the  nerve  substance  extends,  therefore,  for  a  short  dis- 
tance only ;  it  is  strictly  local. 

The  view  that  the  action  of  the  substance  of  the  pounded  nerve 
centres  is  different  from  the  neutralisation  of  the  toxin  by  the  anti- 
toxin of  the  body  fluids  is  further  confirmed  by  the  fact  that  the 
fixation  of  the  tetanus  poison  by  the  cerebral  substance  is  very 
transient.  We  have  shown  that  a  mixture  of  toxin  and  pounded 
cerebral  substance,  that  does  not  produce  any  tetanic  symptom  when 
injected  into  the  peritoneal  cavity  of  guinea-pigs,  gets  up  a  grave 
tetanus  when  it  is  injected  subcutaneously  into  the  thigh.  In  the 

1  Berl  Tdin.  Wchnschr.,  1898,  S.  5. 

2  Deutsche  med.  Wchnschr.,  Leipzig,  1898,  S.  68. 

8  Ann.  de  VInst.  Pasteur,  Paris,  1898,  t.  xn,  p.  91. 

4  Ann.  de  I'lnst.  Pasteur,  Paris,  1898,  t  xn,  pp.  81,  263. 


Artificial  immunity  against  toxins  383 

latter  case  the  toxin  becomes  separated  from  the  particles  of  the  [402] 
cerebral  substance  that  had  fixed  it.  Danysz1  convinced  himself 
that  the  mixture  of  pounded  brain  with  tetanus  toxin  when  it  is 
left  in  physiological  saline  solution,  in  distilled  water,  or  in  a 
10  °/o  solution  of  sea  salt,  allows  the  tetanus  toxin  to  pass  into  the 
macerating  fluid.  The  fixation  of  the  toxin  to  the  cerebral  substance 
is,  therefore,  more  comparable  to  the  mordanting  of  colouring-matters 
by  the  tissues  than  to  a  real  combination. 

Observers  who  have  repeated  the  experiments  of  Wassermann 
and  Takaki  have  been  greatly  struck  by  the  difference  between  the 
action  of  the  pounded  cerebral  substance  and  that  of  the  living  brain 
upon  the  tetanus  toxin.  Whereas  the  former,  taken  from  the  guinea- 
pig,  an  animal  very  susceptible  to  tetanus,  prevented  intoxication 
when  employed  in  minimal  dose,  the  living  brain  of  the  same  species 
was  found  to  be  incapable  of  neutralising  the  most  minute  quantities 
of  toxin.  On  the  other  hand,  Roux  and  Borrel2  have  shown  that  the 
brain  of  rabbits,  whether  untreated  or  vaccinated  against  tetanus, 
was  very  susceptible  to  the  action  of  the  tetanus  toxin.  This  toxin, 
injected  directly  into  the  brain,  set  up  in  both  groups  of  rabbits 
a  special  and  characteristic  cerebral  tetanus.  On  the  other  hand, 
when  a  little  of  the  cerebral  substance  of  the  rabbits,  mixed  in 
vitro  with  tetanus  toxin,  was  injected  into  other  susceptible  animals, 
these  remained  unaffected. 

This  great  difference  between  the  antitoxic  action  of  the  living 
brain  and  that  of  the  pounded  cerebral  matter,  on  the  one  hand, 
and  the  rigorous  localisation  of  the  antitetanic  influence  of  this 
cerebral  substance,  on  the  other,  have  suggested  to  several  observers 
the  idea  that  the  brain  cannot  be  regarded  as  the  organ  of  formation 
of  the  true  antitoxin,  such  as  is  found  in  the  fluids  of  immunised 
animals.  This  view  has  been  expressed  by  Roux  and  Borrel,  Marie 
and  ourselves.  Knorr3  also  shares  this  view,  being  struck  by  the 
fact  that  rabbits  attacked  by  tetanus  remain  for  weeks  with  contrac- 
tions, but  are  incapable  of  producing  in  their  nerve-cells  sufficient 
antitoxin  to  disintoxicate  them,  although  their  blood  is  already 
loaded  with  dissolved  antitoxin. 

At  this  period  it  was  generally  supposed  that,  in  accordance  with 
Ehrlich's  theory,  the  hypothetical  side-chains  were  capable,  under  [403] 

1  Ann.  de  Vlnst.  Pasteur,  Paris,  1899,  t.  xni,  p.  156. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1898,  t.  XII,  p.  225. 

3  Munchen.  med.  Wchnschr.,  1898. 


384  Chapter  XII 

certain  conditions,  not  only  of  fixing  the  tetanus  toxin,  but  also  of 
neutralising  it.  It  was  said,  therefore,  that  these  chains,  reproduced 
in  large  quantities  in  the  cerebral  cells,  must  exercise  their  neutral- 
ising action  in  the  brain  itself.  Consequently,  when  it  was  seen  that, 
in  Roux  and  Borrel's  experiments  on  vaccinated  rabbits,  this  organ 
was  itself  affected,  it  was  concluded  that  the  brain  must  not  be 
regarded  as  the  producer  of  the  antitoxin. 

Later,  Ehrlich  and  his  supporters,  amongst  whom  I  will  name 
especially  Weigert,  have  developed  the  theory  of  side-chains  in  a 
much  more  detailed  fashion,  leading  to  a  different  interpretation  of 
several  facts  previously  established.  Ehrlich  distinguishes  in  the 
toxin  molecule  a  haptophore  group  which  combines  with  the  side- 
chain  or  the  corresponding  receptor  of  the  living  elements,  and  a 
toxophore  group  which  produces  the  poisoning  of  the  protoplasm. 
The  side-chains,  inactive  for  the  toxophore  group,  neutralise  only 
the  haptophore  group.  Consequently,  when  these  side-chains  are 
numerous  in  the  nerve  elements  which  produce  them,  they  may  be 
a  source  of  great  danger  to  this  living  element,  by  attracting  the 
toxic  molecules.  In  this  case,  these  chains,  or  receptors,  serve  to 
attract  the  poison,  just  as  the  badly  adjusted  lightning  conductor 
attracts  lightning.  For  this  reason  rabbits  vaccinated  against  tetanus 
become  tetanic  when  the  toxin  is  injected  directly  into  the  brain. 
It  is  only  at  a  distance  from  the  nerve  centres  that  the  receptors, 
excreted  into  the  body  fluids,  fulfil  their  role  of  true  antitoxins. 
There  they  combine  with  the  haptophore  group  of  the  toxic  molecule, 
leaving  the  toxophore  group  intact ;  this  latter  group,  however, 
diverted  from  the  nerve-cells,  is  incapable  of  exercising  an  injurious 
action. 

From  this  point  of  view  not  only  the  cerebral  tetanus  of  vaccinated 
rabbits,  but  also  the  hypersusceptibility  of  immunised  animals,  upon 
which  von  Behring  has  so  strongly  insisted,  may  be  explained.  The 
argument,  drawn  from  these  facts,  against  the  nervous  origin  of 
tetanus  antitoxin,  loses,  therefore,  much  of  its  weight.  If  we  confront 
this  hypothesis  with  the  other  data  collected  on  the  question,  the 
solution  of  the  problem  becomes  beset  with  great  difficulties.  Previous 
[404]  to  the  discovery  made  by  Wassermann  and  Takaki,  I  attempted  to 
solve  the  problem  by  removing  from  fowls  portions  of  the  brain  and 
spinal  cord,  proposing  to  take  advantage  of  the  fact  that  birds,  which 
are  capable  of  producing  antitoxins,  withstand  these  operations  fairly 
well.  My  hopes  were  not  fulfilled ;  I  could  never  keep  my  fowls 


Artificial  immunity  against  toxins  385 

alive  long  enough  to  complete  the  experiment.  We  must,  therefore, 
for  the  present,  be  content  with  indirect  arguments.  If  the  nerve 
centres  do  really  produce  the  tetanus  antitoxin  and  excrete  it  into 
the  blood,  we  ought  at  a  given  moment  to  find  in  these  organs  a 
greater  quantity  of  this  substance  than  in  the  blood  and  the  other 
organs.  The  reader  will  recall  the  researches  of  Pfeiffer  and  Marx, 
and  of  Deutsch,  who  demonstrated  the  possession  of  a  greater  richness 
in  protective  substance  by  the  phagocytic  organs  of  animals,  treated 
with  micro-organisms,  than  by  the  blood  serum.  The  same  result 
might  be  obtained  by  a  comparative  investigation  of  the  tetanus 
antitoxin  in  the  nerve  centres  and  the  blood  of  animals  immunised 
against  tetanus.  My  experiments  directed  to  this  point  have  not 
been  favourable  to  the  hypothesis  of  the  nervous  origin  of  tetanus 
antitoxin. 

In  fowls,  killed  as  soon  as  tetanus  antitoxin  began  to  appear  in 
the  blood,  the  brain  and  spinal  cord  did  not  exhibit  the  slightest 
antitoxic  power1.  We  might  be  tempted  to  explain  this  result  as  due 
to  an  accumulation  of  toxin  in  the  nerve  centres  which  would  prevent 
the  manifestation  of  the  antitoxin.  For  this  reason,  in  my  later 
researches2,  I  made  use  of  animals  that  had  been  long  immunised, 
but  whose  blood  was  still  antitoxic.  I  killed  a  fowl  which  had  not 
received  any  toxin  for  about  eight  months,  and  a  guinea-pig  into  which 
the  last  toxic  injection  had  been  made  almost  two  years  before  the 
date  of  this  experiment.  After  removing  a  portion  of  the  brain  the 
blood  of  these  two  animals  was  found  to  be  more  antitoxic  than 
before  the  operation,  which  indicated  that  the  source  of  the  antitoxin 
was  as  yet  uninjured.  To  ascertain  whether  this  source  was  to  be 
found  in  the  nerve  centres  I  made  a  comparative  determination  of  the 
antitoxic  power  of  the  brain,  of  the  spinal  cord  and  also  of  several 
other  organs,  of  the  blood  and  of  the  exudations.  The  result  was 
still  negative.  The  nerve  centres  were  found  to  be  less  antitoxic 
than  the  blood  and  other  fluids  of  the  body,  and  even  less  active  than 
such  organs  as  the  liver  and  kidneys. 

In  support  of  the  hypothesis  of  the  nervous  origin  of  tetanus  [405] 
antitoxin  there  remains,  then,  only  the  fact  of  the  retarding  action 
of  the  cerebral  substance  upon  tetanus.     In  the  absence  of  other 
arguments  this  assumes  a  preponderating  importance.     We  have  seen 
that  this  action  is  based  on  a  fleeting  and  not  very  firm  fixation  of  the 

1  Ann.  de  Flnst.  Pasteur,  Paris,  1897,  t.  xi,  p.  801. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1898,  t.  xn,  p.  81. 

B.  25 


386  Chapter  XII 

toxin  by  certain  parts  of  the  brain  and  the  cord.  Are  we  justified  in 
regarding  this  as  comparable  to  the  more  stable  fixation  observed 
in  living  animals  susceptible  to  tetanus  intoxication?  Soon  after 
Wassermann  and  Takaki's  discovery  I  pointed  out  that  the  pounded 
brain  of  frogs  mixed  with  tetanus  toxin  does  not  prevent  animals, 
into  which  this  mixture  is  injected,  from  contracting  fatal  tetanus. 
This  observation  was  confirmed  by  Courmont  and  Doyon1,  in  several 
series  of  experiments  carried  out  under  various  conditions.  They 
found  that "  the  brain  of  the  frog,  heated  or  unheated,  when  mixed 
with  tetanus  toxin  even  for  several  hours,  at  the  temperature  of  the 
laboratory  or  at  38°  C.,  even  in  considerable  doses,  does  not  possess 
any  neutralising  property."  This  fact  would  not  be  in  any  way 
wonderful  if  we  had  to  do  with  an  animal  insusceptible  to  tetanus ; 
but  in  the  frog,  as  we  have  said  in  the  preceding  chapter,  this  is  far 
from  being  the  case.  In  the  cold  it  does  not  readily  become  tetanic, 
but  above  25° — 30°  C.  it  becomes  very  susceptible.  The  turtle,  which 
is  very  refractory  to  this  intoxication,  has  a  brain  which,  when  pounded 
and  mixed  with  tetanus  toxin,  exerts  a  certain  preventive  power  over 
susceptible  animals.  Nevertheless,  the  brain  of  the  living  frog,  as 
demonstrated  by  Morgenroth,  absorbs  this  toxin.  There  is,  therefore, 
a  difference  between  the  absorption  of  the  tetanus  poison  by  the 
living  elements  and  by  the  pounded  cerebral  substance.  A  similar 
result  is  obtained  with  several  other  toxins.  Diphtheria  poison  is 
very  toxic  when  injected  directly  into  the  brain  of  the  guinea-pig  or 
rabbit.  Even  the  rat,  as  demonstrated  by  Roux  and  Borrel2,  is  readily 
affected  by  this  toxin  under  these  conditions.  Doses  which  when 
inoculated  subcutaneously  are  well  borne  by  the  rat,  when  introduced 
into  the  brain  set  up  a  fatal  intoxication  in  this  animal.  And  yet  the 
[406]  brain,  when  pounded  and  mixed  with  diphtheria  toxin,  can  never 
protect  susceptible  animals  from  intoxication.  Numerous  attempts  to 
reproduce  Wassermann  and  Takaki's  experiment  with  the  diphtheria 
poison  have  always  been  unsuccessful.  Attempts  to  obtain  the  same 
result  with  snake  venom  have  also  given  negative  results.  Calmette3 
made  several  experiments  with  emulsions  of  rabbit's  brain  and  snake 
venom  with  the  object  of  ascertaining  whether  the  elements  of  the 
nervous  system  possess  against  venom  the  same  properties  as  against 
tetanus  toxin.  "None  of  these  emulsions" — concludes  Calmette — 
"  exhibited  either  the  slightest  protective  or  antitoxic  power  in  vitro. 

1  Compt.  rend.  Soc.  de  biol.,  Paris,  1898,  p.  602. 
*  Ann.  de  Vlnst.  Pasteur,  Paris,  1898,  t.  xn,  p.  238.  3  Ic.  p.  343. 


Artificial  immuitity  against  toxins  387 

There  is,  therefore,  no  analogy  of  action  between  what  takes  place  in 
the  nerve  elements  against  tetanus  toxin  and  against  venom."  Never- 
theless venom,  like  diphtheria  toxin  and  tetanus  toxin  in  the  frog, 
exerts  an  undoubted  action  on  the  nerve  centres. 

Again,  the  protective  fixation  of  poisons  to  the  cerebral  sub- 
stance is  not  the  exclusive  privilege  of  tetanus  toxin.  Kempner  and 
Schepilewsky1  obtained  the  same  result  with  the  toxin  of  botulism 
(produced  by  van  Ermenghem's  anaerobic  micro-organism  which  sets 
up  intoxication  of  intestinal  origin  in  certain  cases  of  poisoning  by 
food).  The  brain  and  spinal  cord  of  the  guinea-pig,  when  triturated 
with  physiological  salt  solution  and  mixed  with  botuliuic  toxin, 
prevents  intoxication  in  susceptible  animals,  exactly  as  in  Wassermann 
and  Takaki's  experiments  with  tetanus. 

When  Kempner  and  Schepilewsky  wished  to  obtain  some  idea  as 
to  the  substance  or  substances  in  the  nerve  centres  which  fix  the 
toxin  of  botulism  and  thus  prevent  poisoning,  they  found  that  lecithin 
and  cholesterin,  mixed  with  this  toxin  or  injected  separately  and 
simultaneously,  protected  mice  just  as  completely  as  did  the  cerebral 
substance.  On  the  other  hand,  they  found  a  difference  as  regards  the 
two  substances  when  injected  before  the  toxin  was  introduced  ;  they 
were  then  unable  to  prevent  poisoning,  though  the  cerebral  substance 
exerted  an  undoubted  protective  influence.  Kempner  and  Schepilew- 
sky also  showed  that  heating  altered  the  preventive  action  of  lecithin 
and  cholesterin  less  than  it  did  that  of  cerebral  emulsion. 

These  observers  extended  their  researches  to  the  protective  action  [407] 
of  fats  and  demonstrated  that  olive  oil  when  emulsified  and  neutra- 
lised with  soda  and  mixed  with  twice  and  even  four  times  the  lethal 
dose  of  botuliuic  toxin,  prevented  the  contraction  of  a  fatal  poisoning 
by  mice.  Tyrosin  also  protected  mice  against  this  intoxication,  not 
only  when  injected  simultaneously  with  the  poison,  but  even  when 
introduced  into  the  animal  24  hours  before  the  poison  was  ad- 
ministered. Kempner  and  Schepilewsky  conclude  "that  not  only 
with  the  substance  of  the  nerve  centres,  but  also  with  various  other 
substances,  they  were  able  to  obtain  a  certain  protective  effect  against 
the  toxin  of  botulism"  (p.  221).  Their  experiments  with  cholesterin 
and  tyrosin  were  suggested  to  them  by  the  previous  researches  of 
Phisalix2  who  demonstrated  that  the  bile  salts,  as  well  as  the  two 

1  Ztschr.f.  Hyg.,  Leipzig,  1898,  Bd.  xxvn,  S.  213. 

*  Compt.  rend.  Acad.  d.  sc.,  Paris,  1897,  p.  1053;  and  1898,  p.  431 ;  Compt.  rend. 
Soc.  de  Uol,  Paris,  1897,  p.  1057 ;  and  1898,  p.  153. 

25—2 


388  Chapter  XII 

substances  I  have  just  mentioned,  would  protect  animals  against  the 
venom  of  the  viper. 

Bearing  all  these  facts  in  mind,  it  appears  to  be  probable  that  in 
the  above  cases  it  is  principally  the  fatty  matters  of  the  nerve  centres 
that  temporarily  fix  these  toxins,  and  allow  the  animal  organism  to 
divert  the  poisons  from  their  morbific  action.  From  this  point  of 
view,  it  is  interesting  to  note  that  the  toxic  action  of  the  tetanus 
poison  can  also  be  prevented  by  other  substances  than  the  emulsion 
of  the  nerve  centres.  Thus  Stoudensky1  demonstrated,  in  an  investi- 
gation carried  out  in  Roux's  laboratory,  that  carmine  fixes  the 
tetanus  toxin  and  prevents  its  action  on  the  guinea-pig.  As  in  the 
case  of  the  cerebral  substance,  this  fixation  by  carmine  is  very 
unstable.  When  the  carmine  that  has  fixed  the  tetanotoxin  is 
macerated  in  distilled  water  it  gives  up  the  poison  to  the  water  which 
is  then  capable  of  producing  tetanus.  Such  fixation  does  not  end,  any 
more  than  in  the  case  of  the  cerebral  substance,  in  the  destruction  or 
disappearance  of  the  toxin.  Carmine  if  first  dissolved  or  macerated 
in  water  (especially  if  heated)  loses  its  fixative  power  and  can  no 
longer  prevent  tetanus  poisoning.  Sterilisation,  at  120°,  100°  and 
even  at  60°  C.,  of  the  carmine,  suspended  in  physiological  salt 
solution,  caused  it  to  lose  its  protective  action,  although  dry  heat 
applied  to  it  in  closed  tubes  did  not  destroy  this  power. 
[408]  In  many  respects  carmine,  which  is  derived  especially  from  the 
adipose  body  of  the  cochineal  insect,  exerts  an  antitoxic  influence 
analogous  to  that  of  maceration  with  the  nerve  centres.  If  fats  play 
a  special  part  in  this  action,  we  can  readily  understand  how  a  brain, 
such  as  that  of  the  frog,  poor  in  fatty  matters,  cannot  fix  the  tetanus 
toxin  and  prevent  its  morbific  action.  In  any  case  the  fact  that 
certain  substances  of  diverse  nature,  acting  on  toxins,  exert  an 
influence  similar  to  that  of  the  pounded  mass  of  the  nerve  centres, 
does  not  allow  us  to  accept  Wassermann  and  Takaki's  experiment  as 
proving  the  nervous  origin  of  tetanus  antitoxin.  The  analogy  with 
the  facts  bearing  on  the  anticytotoxins,  collected  and  described  in  the 
fifth  chapter,  also  tells  against  this  hypothesis.  We  would  here  remind 
the  reader  that  the  two  constituent  parts  of  the  antispermotoxin, 
the  anticytase  and  the  antispermofixative,  develop  in  castrated 
animals  and  are  consequently  produced  outside  the  spermatozoa, 
elements  susceptible  to  the  spermotoxin.  The  facts  collected  con- 

1  Ann.  de  I'lnst.  Pasteur,  Paris,  1899,  t.  xni,  p.  126. 


Artificial  immunity  against  toxins  389 

cerning  the  antihaemotoxins  indicate  also  that  these  substances  have 
some  other  origin  than  the  red  blood  corpuscles. 

This  latter  supposition  appears  to  be  in  contradiction  to  Ransom's1 
very  interesting  researches  on  the  haemolytic  action  of  saponin, 
carried  out  in  Meyer's  laboratory  at  Marburg.  This  glucoside,  owing 
to  its  property  of  fixing  itself  on  the  stroma  of  these  corpuscles 
dissolves  the  red  corpuscles  of  many  vertebrates.  The  cholesterin  of 
this  stroma  combines  with  the  saponin,  as  the  result  of  which  the  red 
corpuscles  become  altered  and  allow  the  haemoglobin  to  diffuse.  But 
this  same  substance,  cholesterin,  which  causes  the  poison  to  penetrate 
into  the  red  blood  corpuscles,  prevents  the  solution  of  these  elements 
when  they  are  bathed  in  blood-serum.  This  fluid,  in  fact,  acts  as  the 
antitoxin  to  saponin  and  does  so  just  because  it  contains  cholesterin. 
The  cholesterin  of  the  serum,  fixing  the  saponin,  prevents  it  from 
affecting  the  red  corpuscles,  thus  fulfilling  the  function  of  a  well 
fitted  lightning  conductor.  On  the  other  hand,  when  the  cholesterin 
of  the  stroma  of  these  corpuscles  is  linked  on  to  the  saponin,  it 
renders  them  the  disservice  of  a  defective  lightning  conductor.  The 
accord  between  these  facts  and  the  postulates  of  Ehrlich's  theory  led 
Ransom  to  suppose  that  in  the  haemolysins  and  antihaemolysins,  [409] 
cholesterin  perhaps  played  a  similar  part.  His  experiments  con- 
vinced him  that  this  was  not  the  case.  As  it  is  generally  accepted, 
after  Calmette's2  experiments  and  according  to  Ehrlich's  view,  that 
the  alkaloids  and  the  glucosides  in  general  are  incapable  of  setting  up 
the  formation  of  antitoxins,  we  might  regard  the  attempts  to  find  an 
antisaponin  and  to  settle  whether  it  is  identical  with  cholesterin  as 
useless.  But  in  regard  to  these  delicate  questions  we  must  be  careful 
not  to  give  too  great  weight  to  a  priori  arguments.  It  was  believed 
until  quite  recently  that  substances  with  very  complex  molecules, 
such  as  the  albuminoids,  toxins  and  soluble  ferments,  must  always 
give  rise  to  the  production  of  antibodies  in  the  animal ;  whilst  the 
simpler  substances  whose  chemical  nature  was  better  defined  could 
never  lead  to  this.  Facts  acquired  in  recent  years  have  led  to  a 
modification  of  this  view.  In  our  fifth  chapter  we  have  already  spoken 
of  the  fruitless  attempts  of  Ehrlich  and  Morgenroth  to  obtain  certain 
antifixatives.  And  yet  the  fixatives,  as  is  shown  by  the  results  of 
the  researches  of  Bordet  and  myself,  belong  to  the  category  of 
substances  which  are  quite  capable  of  setting  up  the  formation  of 

1  Deutsche  med.  Wchnschr.,  Leipzig,  1901,  S.  194. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p,  244. 


390  Chapter  XII 

antibodies.  Again,  certain  mineral  poisons,  quite  unexpectedly,  gave 
rise  to  the  development  of  the  counter-poison  in  the  animal  body. 
This  feet  forced  itself  upon  Besredka1  in  his  researches  on  the 
adaptation  to  arsenic  made  in  my  laboratory.  His  experiments  were 
undertaken  for  the  purpose  of  studying  the  mechanism  of  the  re- 
fractory condition  against  a  poison,  apart  from  any  antitoxic  action 
whatever,  which,  according  to  previous  investigations,  seemed  excluded. 
This  action,  however,  was  exhibited  in  such  a  degree  that  it  could  not 
be  ignored.  The  serum  of  animals  immunised  against  arsenious 
acid  was  found  to  possess  both  protective  and  antitoxic  properties 
against  a  dose  of  this  poison  killing  a  rabbit  in  48  hours.  It  is  true 
that  Morishima2,  in  a  research  carried  out  in  Hey  man's  laboratory  at 
Ghent,  has  thrown  doubt  upon  these  results.  His  objections,  how- 
[410]  ever,  cannot  refute  the  statements  of  Besredka  which  rest  on  very 
precise  and  numerous  experiments  which  I  witnessed.  Morishima 
left  out  of  account  several  important  circumstances  and  carried  out 
his  experiments  without  any  continuous  check  by  means  of  control 
animals.  It  must  be  said  also  that  the  resistance  of  the  rabbit 
against  arsenic  depends  on  many  different  factors  and  that,  at  certain 
seasons,  it  is  much  more  difficult  to  adapt  them  to  the  poison  than  at 
others.  It  is  only  by  numerous  researches  extending  over  a  very  long 
period  that  we  can  arrive  at  precise  and  conclusive  results. 

From  these  observations  there  is  every  inducement  for  us  to 
attempt  to  ascertain  whether,  by  subjecting  animals  to  repeated 
injections  of  saponin,  it  is  possible  to  augment  the  antisaponic  power 
of  their  blood-serum  and  whether,  if  this  takes  place,  the  antitoxic 
action  is  due  to  a  rise  in  the  amount  of  cholesterin  in  this  serum.  I 
therefore  requested  Besredka  to  carry  out  some  experiments  bearing 
on  this  point.  Guinea-pigs,  injected  with  progressive  doses  of  saponin 
for  more  than  two  months,  at  the  end  of  this  period  showed  no 
increase  in  the  antisaponic  power  of  their  serum.  They  followed  the 
rule  established  by  Ehrlich  ;  they  developed  no  antitoxin  against  a 
glucoside.  Moreover,  they  gave  us  no  new  information  as  to  the 
origin  of  these  antibodies. 

In  his  first  memoir  in  which  the  theory  of  side-chains  is  treated, 
Ehrlich  insists  on  the  nervous  origin  of  antitetanin  as  an  example  of 
the  production  of  antitoxins  by  animals  susceptible  to  poisons.  Now, 
however,  that  he  has  come  to  distinguish  haptophore  and  toxophore 

1  Ann.  de  FInst.  Pasteur,  Paris,  1899,  t.  xm,  p.  465. 

2  Arch,  internal,  de  Ptiarmacodyn.,  Gaud  et  Paris,  1900,  vol.  vn,  p.  65. 


Artificial  immunity  against  toxins  391 

groups  in  the  toxic  molecule,  it  is  to  the  side-chain,  which  fixes  the 
first  group,  that  Ehrlich  attributes  prime  importance.  "  The  forma- 
tion of  antitoxins  " — he  says1  in  the  opening  address  at  his  Institute 
at  Frankfort — "  would,  therefore,  be  absolutely  independent  of  the 
action  of  the  toxophore  elements."  In  other  words,  for  a  cell  to  be 
capable  of  producing  antitoxin,  it  is  not  at  all  necessary  that  it  should 
be  susceptible  to  the  toxic  influence  of  the  poison ;  it  is  only  necessary 
that  it  should  possess  receptors,  or  side-chains,  capable  of  combining 
with  the  haptophore  group  of  the  toxin.  Thus  it  is  possible,  as  we 
have  described  above,  to  produce  antitoxins,  with  modified  toxins  [41 1] 
whose  toxic  action  is  nil  or  almost  so,  but  which  have  retained  their 
power  of  combining  with  antitoxic  substances.  According  to  Ehrlich, 
these  modified  toxins  are  toxoids,  in  which  the  toxophore  group  is 
completely  destroyed;  "whilst  the  haptophore  group,  the  producer  of 
immunising  substances,  is  retained  in  its  integrity."  It  is  evident 
then  that,  under  such  conditions,  the  tetanus  antitoxin  might  be 
developed  elsewhere  than  in  the  nerve  centres.  For  that  it  would  be 
sufficient  that  outside  the  nerve  cells  there  should  be  other  living 
elements  capable  of  fixing  the  tetanus  toxin,  or,  to  use  Ehrlich 's 
phraseology,  elements,  possessing  side-chains,  having  an  affinity  for 
the  haptophore  group  of  the  tetanus  poison. 

Doiiitz2  has  already  expressed  the  view  that  in  the  rabbit  the 
tetanus  toxin  may  be  fixed  not  only  by  the  nerve  elements  but  also 
by  the  various  other  cells. 

The  existence  of  such  cells,  outside  the  nervous  system,  is  not 
merely  hypothetical.  It  is  shown  very  clearly  in  Roux  and  Sorrel's 
experiments  on  cerebral  tetanus.  In  order  to  produce  this  disease  in 
the  rabbit,  it  is  sufficient  to  introduce  a  very  small  dose  of  toxin 
directly  into  the  brain.  When  inoculated  subcutaneously  with  much 
larger  quantities  of  the  same  tetanus  poison,  the  rabbit  remains  in 
good  health  or  exhibits  merely  a  slight  and  transient  tetanus.  "  The 
resistance  of  the  rabbit  against  the  tetanus  toxin,  injected  under  the 
usual  conditions" — conclude  Roux  and  Borrel3 — "is  not  due,  then, 
to  a  relative  insusceptibility  of  the  nerve  centres,  but  to  the  fact 
that  much  of  the  poison  introduced  does  not  reach  the  nerve  cells 
and  is  destroyed  in  some  part  of  the  animal."  In  the  guinea-pig, 
as  shown  by  the  same  investigators,  the  difference  of  the  dose  of 
tetanus  poison,  necessary  to  produce  fatal  tetanus  by  intracerebral  or 

1  Semaine  med..  Paris,  1899,  p.  411. 
2  Deutsche  mecl.  Wchnschr.,  Leipzig,  1897,  S.  428.  3  i.e.  p.  229. 


392  Chapter  XII 

by  subcutaneous  injection,  is  minimal  or  nil,  from  which  it  may  be 
argued  that  in  this  very  susceptible  animal  there  is  no  destruction 
of  toxin  outside  the  nerve  centres  and  that  the  whole  of  the  poison 
introduced  makes  its  way  without  hindrance  as  far  as  these  organs. 
Ehrlich,  in  his  report  to  the  International  Congress  of  Medicine  in 
Paris  (August,  1900),  accepted  these  results,  as  seen  from  his  tenth 
[412]  and  eleventh  propositions :  "  The  receptors  exist,  sometimes  in  certain 
tissues  only,  sometimes  in  the  majority  of  the  organs  (action  of 
tetanus  poison  in  the  guinea-pig  and  in  the  rabbit),"  "  ...the  presence 
of  numerous  receptors  in  the  organs  of  less  vital  importance  may 
bring  about — thanks  to  a  kind  of  diversion  of  the  toxin  molecules — a 
diminution  in  the  susceptibility  of  the  animal  to  this  toxin1."  We  must 
here  recall  the  differences  between  the  susceptibility  of  the  guinea- 
pig  and  that  of  the  rabbit  to  small  doses  of  tetanus  toxin  frequently 
repeated  as  in  Knorr's  experiments  already  referred  to.  The  guinea- 
pig,  subjected  to  these  injections,  dies  in  a  tetanic  condition  long 
before  it  has  received  the  minimal  lethal  dose  for  this  species  when 
injected  in  a  single  dose.  The  rabbit,  on  the  other  hand,  is  very 
tolerant  of  repeated  doses  and  even  rapidly  acquires  an  immunity 
against  five  minimal  lethal  doses  for  the  rabbit  (injected  at  once). 
Knorr  explained  this  difference  as  due  to  the  hypersusceptibility 
of  the  nerve  centres  in  the  guinea-pig  and  to  their  acquired  in- 
susceptibility in  the  rabbit.  The  experiments  of  Roux  and  Borrel 
on  the  cerebral  tetanus  of  rabbits  vaccinated  against  tetanus,  have 
demonstrated  that  this  insusceptibility  is  not  produced  in  these 
animals.  We  must,  therefore,  seek  some  other  explanation.  In 
rabbits  subjected  to  small  repeated  doses,  the  poison  is  more  and 
more  prevented  by  certain  living  elements  from  reaching  the  nerve 
centres.  Further,  it  is  neutralised  by  the  antitoxin  which  is  rapidly 
produced.  We  find  from  Knorr's2  researches  that  in  rabbits  anti- 
toxin appears  in  the  blood  in  cases  where,  affected  with  a  transitory 
tetanus,  their  limbs  remain  contracted  for  weeks.  In  guinea-pigs, 
affected  with  the  same  form  of  tetanus,  antitoxin  in  appreciable 
quantity  is  never  found,  even  after  complete  recovery.  All  these 
facts  accord  with  the  hypothesis  that  there  exist,  outside  the  nervous 
system,  certain  living  cells  which  absorb  the  tetanus  toxin  and  pro- 
duce antitoxin.  Rabbits  and  fowls  possess  this  property  in  a  much 

1  Compt.  rend.  Congres  intemat.  de  Medicine  de  Paris,  Section  de  bacteriologie 
et  de  parasitologie,  Paris,  1891,  p.  30. 

2  Munchen.  med.  Wchnschr.,  1898,  S.  321. 


Artificial  immunity  against  toxins  393 

greater  degree  than  do  guinea-pigs.  The  fowl,  according  to  Knorr, 
develops  "  a  large  quantity  of  antitoxin,  whilst  the  tetanic  symptoms 
are  still  augmenting."  In  this  animal,  as  we  have  been  able  to  show1, 
a  portion  of  the  tetanus  toxin  is  absorbed  by  the  leucocytes.  By  [413] 
exciting  aseptic  exudations  in  fowls  into  which  I  had  previously 
injected  this  toxin,  I  was  able  to  convince  myself  that  these  exuda- 
tions, much  richer  in  leucocytes  than  was  the  blood,  were  also  much 
more  tetanigenic  than  was  the  blood.  I  observed  also  a  more  or 
less  pronounced  leucocytosis  after  the  injection  of  non-lethal  doses 
of  tetanus  toxin  into  fowls.  It  is  possible  that  the  leucocytes  were 
actual  agents  in  protecting  the  animal  against  the  penetration  of  this 
poison  to  the  susceptible  nerve  centres. 

The  great  susceptibility  of  leucocytes  to  microbial  toxins  serves 
to  indicate  that  these  cells  are  of  some  importance  in  the  struggle 
of  the  animal  against  these  poisons.  Their  injection  usually  sets  up 
a  marked  hyperleucocytosis  of  the  blood.  On  this  point  Chatenay2, 
working  in  my  laboratory,  has  carried  out  a  series  of  experiments  on 
animals  poisoned  by  bacterial  (tetanus  and  diphtheria),  phanero- 
gamic (ricin  and  abrin)  and  animal  (snake  venom)  toxins.  He  was 
able  to  demonstrate  a  striking  analogy  between  them  and  the  pheno- 
mena which  occur  in  bacterial  infections.  When  death  takes  place 
at  the  end  of  a  very  short  period,  the  number  of  leucocytes  markedly 
diminishes ;  if  the  animal  lives  beyond  24  hours  or  resists  completely, 
a  hyperleucocytosis,  often  of  very  marked  character,  is  produced.  In 
the  guinea-pig,  which  is  so  susceptible  to  tetanus,  the  leucocytosis 
observed  occurs  even  after  injections  of  quantities  of  tetanus  toxin 
equal  to  several  lethal  doses,  and  it  is  only  after  the  introduction  of 
an  amount  equal  to  one  hundred  times  the  lethal  dose  that  the 
number  of  leucocytes  remains  stationary  or  shows  a  diminution. 
Here  we  have  something  analogous  to  what  takes  place  against  the 
anthrax  bacillus  in  the  same  animal.  The  penetration  of  this  deadly 
organism  sets  up  a  marked  leucocytosis,  but  the  accumulated  leuco- 
cytes are  incapable  of  seizing  the  bacilli  or  of  preventing  their  noxious 
action.  In  other  species  of  animals,  such  as  the  rabbit  and  the  fowl, 
the  intervention  of  the  leucocytes  against  the  anthrax  bacillus,  as 
well  as  against  the  tetanus  toxin,  is  more  effective. 

If  this  toxin,  instead  of  being  injected  in  solution,  be  introduced 
along  with  the  bodies  of  the  micro-organisms  which  contain  it,  the 

1  Ann.  de  Vlnst.  Pasteur,  Paris,  1897,  t.  XT,  p.  808. 

2  "  Les  reactions  leucocytaires,  vis-a-vis  de  certaines  toxines,"  Paris,  1894. 


894  Chapter  XII 

struggle  on  the  part  of  the  animal  takes  place  under  more  favourable 
conditions  and  even  very  susceptible  animals  may  afford  evidence 
[414]  that  they  offer  a  high  resistance.  Vaillard  and  Vincent1  have 
shown  that  if  we  inject  living  tetanus  bacilli,  or  the  spores  of  these 
bacilli,  deprived  of  free  toxin,  into  guinea-pigs  a  great  accumulation 
of  leucocytes,  which  prevent  the  production  of  infection  and  intoxi- 
cation by  devouring  the  bacilli  and  their  spores,  takes  place.  The 
toxin  contained  in  the  ingested  bacilli  remains  innocuous ;  this  afford- 
ing evidence  of  the  protective  part  played  by  the  leucocytes  against 
the  toxin.  The  same  interpretation  may  be  offered  to  explain  the 
survival  of  animals  very  susceptible  to  tetanus,  when  the  tetanus 
poison,  mixed  with  pounded  cerebral  substance  or  with  carmine 
powder,  is  injected.  In  these  mixtures  the  toxin,  as  mentioned  above, 
becomes  attached  to  certain  substances  of  the  triturated  brain  or  to 
the  grains  of  carmine.  This  fixation  is  very  unstable,  the  toxin  is 
readily  set  free  j-'butj"  Vhen  introduced  into  the  body  of  the  animal, 
the  mixture  induces  a  great  accumulation  of  leucocytes  which  seize 
the  cerebral  particles  and  the  grains  of  carmine  and  along  with  them 
take  possession  of  the  toxin.  Wassermann  and  Takaki's  experiments 
and  those  of  Stoudensky  are  easily  explained  if  we  assume  two 
protective  acts :  the  first  of  these  consists  in  fixing  the  toxin,  thus 
preventing  it  from  diffusing  and  rapidly  reaching  the  living  nerve 
cells ;  the  second  is  the  absorption  of  the  toxin  fixed  by  the  leuco- 
cytes,— cells  endowed  with  receptors  for  the  haptophore  group  of 
the  toxin,  but  insusceptible  to  its  toxophore  group.  When  one 
of  the  two  factors  is  absent,  tetanus  cannot  be  prevented.  It  is  for 
this  reason  that  in  Courmont  and  Doyon's  experiments  with  emulsion 
of  the  frog's  brain,  mixed  with  tetanus  toxin,  the  inoculated  animals 
died  from  tetanus  in  spite  of  an  accumulation  of  leucocytes.  This 
fact  affords  additional  proof  that,  under  these  conditions,  the  toxin 
does  not  become  anchored  to  the  particles  of  the  pounded  cerebral 
substance,  this  anchoring  being  a  condition  necessary  for  the  effective 
reaction  of  the  leucocytes. 

The  absorption  of  the  tetanus  toxin  becomes  evident  when  we 
study  in  detail  the  phenomena  produced  in  the  experiments  carried 
out  according  to  Vaillard's  methods  with  tetanus  spores  and  those 
of  Wassermann  and  Takaki  with  poison  to  which  cerebral  emulsion 
has  been  added,  or  according  to  Stoudensky's  method  with  grains  of 
carmine.  When,  however,  it  is  desired  to  bring  forward  rigorous 
1  Ann.  de  Flint.  Pasteur,  Paris,  1891,  t.  v,  p.  1. 


Artificial  immunity  against  toxins  395 

proof  of  the  presence  of  the  tetanus  toxin  inside  the  leucocytes  [415] 
charged  with  spores,  with  granules  of  cerebral  substance  or  with 
grains  of  carmine,  very  great  difficulties  are  encountered.  How, 
indeed,  is  it  possible  to  demonstrate  this  poison  fixed  upon  these 
various  bodies,  a  poison,  the  presence  of  which  cannot  be  demon- 
strated except  by  its  injection  into  the  animal  ?  For  this,  in  the  study 
of  the  reaction  of  the  organism  of  the  animal  against  the  poisons,  it 
is  very  important  to  have  recourse  to  substances,  whose  presence  can 
be  demonstrated  more  easily  than  can  the  microbial  toxins.  We  must 
first  have  recourse  to  the  alkaloids,  especially  atropin,  which,  in  this 
respect,  present  numerous  advantages.  We  know  that  rabbits  resist 
considerable  doses  of  sulphate  of  atropin,  even  when  this  poison  is 
injected  directly  into  the  blood.  On  the  other  hand,  when  it  is 
introduced  into  the  brain,  according  to  Roux  and  Borrel's  method, 
even  small  quantities  are  quite  sufficient,  as  demonstrated  by  Cal- 
mette1,  to  produce  a  fatal  poisoning.  The  intracerebral  injection 
of  the  one-hundredth  part  of  a  dose  which,  when  introduced  into  the 
circulation  of  the  rabbit,  produces  no  disturbance,  in  the  same 
animal  at  the  end  of  a  few  minutes  sets  up  an  enormous  pupillary 
dilatation  with  symptoms  of  very  lively  excitation,  increase  of  the 
reflexes,  and  general  anaesthesia.  These  phenomena  are  succeeded 
by  paralysis  and  death,  which  supervenes  three  or  four  hours  after 
the  injection.  The  natural  immunity  of  the  rabbit  against  atropin 
falls  therefore  into  the  same  category  as  that  against  morphin.  It 
is  not  due  to  the  innate  insusceptibility  of  the  nerve  cells,  but  to 
something  which  prevents  the  alkaloid  from  reaching  these  living 
elements.  With  the  object  of  ascertaining  the  mechanism  of  this 
immunity,  Calmette  injected  into  the  veins  of  rabbits  a  fairly  large 
quantity  of  sulphate  of  atropin  (0*2),  he  then  bled  these  animals  and 
collected  from  their  blood  the  plasma  and  the  white  corpuscles, 
separating  them  by  centrifugalisation.  When  injected  into  the  brain 
of  other  rabbits,  these  constituents  of  the  blood  did  not  act  in  the 
same  way.  Whilst  large  doses  of  plasma  set  up  merely  a  short  period 
of  excitation  and  a  very  transitory  pupillary  dilatation,  corresponding 
quantities  of  leucocytes  caused  grave  disturbances,  sometimes  followed 
by  death  in  from  seven  to  twelve  hours.  Calmette  concludes  from  his 
researches  that  the  atropin  does  not  remain  in  the  fluid  part  of  the 
blood,  since  mere  traces  of  it  are  found  in  the  serum,  but  that  it  is 

1  "  Cinquantenaire  de  la  Societe  de   Biologic,"  Volume  jubilaire,  Paris,  189.9, 
p.  202. 


396  Chapter  XII 

[416]  seized  and  absorbed  almost  immediately  by  the  leucocytes1.  This 
result  has  been  confirmed  by  Lombard2  by  another  series  of  experi- 
ments. After  injecting  very  large  quantities  of  sulphate  of  atropin 
into  rabbits  and  guinea-pigs,  he  bled  these  animals  and  separated 
out  the  elements  of  their  blood.  Instead  of  introducing  these 
elements  into  the  brain  of  rabbits,  he  injected  them  into  cats, 
animals  very  sensitive  to  atropin.  The  cats  which  received  the  red 
corpuscles  and  the  plasma  exhibited  very  insignificant  symptoms  of 
poisoning.  Those,  on  the  other  hand,  which  were  injected  with  a 
corresponding  quantity  of  leucocytes,  had  much  graver  symptoms  of 
intoxication,  such  as  photophobia  with  maximal  pupillary  dilatation, 
dysphagia  and  persistent  diarrhoea. 

It  is,  therefore,  to  the  absorption  of  the  atropin  by  the  leucocytes 
that  naturally  refractory  animals  owe  their  immunity,  an  immunity 
which  is  very  marked  in  spite  of  the  susceptibility  of  the  nervous 
elements  of  these  animals.  We  have  been  able  to  obtain  this  result 
thanks  to  the  delicate  physiological  reactions  obtained  with  certain 
alkaloids.  As  regards  arsenic  the  demonstration  could  be  pushed 
even  further;  for  the  absorption  of  this  mineral  poison  by  the 
leucocytes  has  been  established  by  chemical  analysis. 

When  engaged  in  my  researches  on  the  leucocytic  phenomena  in 
intoxications  I  succeeded3  in  showing  that  in  rabbits  subjected  to 
rapidly  fatal  doses  of  arsenious  acid,  there  is  a  marked  diminution  in 
the  number  of  white  corpuscles  in  the  blood.  On  the  other  hand, 
in  rabbits  habituated  to  arsenic,  the  same  doses  which  brought  about 
hypoleucocytosis  and  death  of  the  control  rabbits,  induced  a  con- 
siderable rise  in  the  number  of  leucocytes.  Later,  Besredka4  made 
continuous  and  detailed  researches  upon  this  subject  and  obtained 
most  interesting  results.  In  order  to  simplify  the  conditions  of 
experiment,  he  studied  the  reaction  of  the  organism  of  the  animal 
[417]  after  the  introduction  of  a  red  trisulphide  of  arsenic5,  a  not  very 

1  The  rapid  disappearance  of  poisons  from  the  blood  is  proved  also  by  the  ex- 
periments of  von  Behring,  Donitz,  Decroly  and  Rousse  (Arch,  internat.  de  Pharma- 
codyn.,  Gand  et  Paris,  1899,  t.  vi,  p.  211)  on  snake  venom  and  diphtheria  and  tetanus 
toxins,  as  likewise  by  those  of  Heymans  and  Masoin  (Ibid.,  1901,  t.  vm,  p.  1)  on  the 
malonic  and  pyrotartaric  nitrites.    These  poisons,  within  a  few  minutes  of  their 
injection  into  the  veins,  are  absorbed  by  the  cell  elements. 

2  "  Contribution  a  1'etude  physiologique  du  leucocyte,"  Paris,  1901,  p.  39. 

3  Ann.  de  FInst.  Pasteur,  Paris,  1894,  t.  vm,  p.  719. 

4  Ann.  de  I'Inst.  Pasteur,  Paris,  1899,  t.  xm,  pp.  49,  209. 

6  See  Besredka,  op.  cit.,  p.  50,  for  its  approximate  composition  and  distinction 
from  ordinary  yellow  trisulphide. 


Artificial  immunity  against  toxins  397 

soluble  salt,  easily  recognisable  by  its  colour  and  markedly  toxic. 
When  non-lethal  doses  of  this  salt  were  injected  into  the  peritoneal 
cavity  of  the  guinea-pig,  there  was,  first  a  transitory  fall  in  the 
number  of  the  white  corpuscles  in  the  peritoneal  fluid,  followed  by  a 
hyperleucocytosis  of  the  most  marked  character.  Of  the  leucocytes 
accumulated  in  the  exudation  the  macrophages  almost  exclusively 
seized  the  yellowish-red  granules  of  the  trisulphide  of  arsenic. 
Very  shortly,  the  whole  of  the  salt  injected  was  found  within  the 
peritoneal  leucocytes,  and  the  animals  in  which  this  marked  phago- 
cytosis occurred  remained  in  good  health.  The  ingested  granules 
could  be  observed  for  several  days  in  the  macrophages ;  but  in 
course  of  time,  these  arsenical  particles  were  broken  up  into  very 
small  granules  and  ultimately  disappeared.  Here,  then,  we  have  an 
intraphagocytic  solution  of  the  trisulphide  of  arsenic  and  very  pro- 
bably a  transformation  of  this  salt  into  some  other  arsenical  combina- 
tion, innocuous  to  the  animal.  This  soluble  substance  escapes  from 
the  macrophages  and  is  finally  excreted  by  the  urinary  passages. 

Since  the  phagocytes  ingest  the  trisulphide  of  arsenic  and  render 
it  innocuous,  it  was  to  be  anticipated  that  the  elimination  of  these 
protective  cells  would  lead  to  a  fatal  poisoning  by  doses  which,  under 
normal  conditions,  are  readily  withstood  by  guinea-pigs.  When 
Besredka  used  sacs  of  reed-pith  containing  non-fatal  quantities  of 
the  red  trisulphide  and  introduced  them  into  the  peritoneal  cavity 
of  guinea-pigs  these  animals  were  not  long  in  exhibiting  symptoms 
of  poisoning  and  died  at  the  end  of  a  longer  or  shorter  period,  this 
varying  with  the  amount  of  poison  introduced.  Even  when  the 
phagocytic  reaction  had  been  impaired  as  the  result  of  a  previous 
injection  of  carmine  powder,  the  guinea-pigs  died  after  doses  of 
trisulphide  of  arsenic  which,  under  ordinary  conditions,  did  not  kill 
them.  The  phagocytes  in  this  experiment  devoured  numerous  grains 
of  carmine  and  were  rendered  incapable  of  ingesting  enough  of  the 
trisulphide  of  arsenic  to  save  the  animal.  On  the  other  hand,  when 
Besredka  set  up  a  previous  accumulation  of  macrophages  in  the 
peritoneal  cavity  of  his  guinea-pigs,  he  succeeded  in  rendering 
these  animals  resistant  to  doses  of  trisulphide  of  arsenic  that,  under 
normal  conditions,  were  fatal.  The  whole  of  these  facts  converge 
to  show  that  the  phagocytes,  thanks  to  their  power  of  seizing  the 
trisulphide  of  arsenic  and  of  modifying  it  within  them,  exercise  a 
beneficent  and  immunising  action  on  the  organism  of  the  animal.  [418] 
The  analogy  of  the  main  facts  concerning  this  protective  influence 


398  Chapter  XII 

with  that  observed  in  the  immunity  against  infective  micro-organisms 
is  indeed  very  considerable. 

Having  determined  the  part  played  by  the  macrophages  in  the 
resistance  of  the  organism  of  the  animal  against  a  not  very  soluble 
salt  of  arsenic,  Besredka  proceeded  to  study  the  leucocytic  phenomena 
in  poisoning  by  soluble  arsenical  compounds.  In  his  experiments 
he  made  use  of  potassium  arsenite  and  he  found  that  when  lethal 
doses  were  injected  the  guinea-pigs  showed  a  diminution  of  leuco- 
cytes in  the  blood  in  less  than  24  hours,  whilst  with  non-lethal  doses, 
he  produced  a  marked  hyperleucocytosis.  When  he  injected  lethal 
doses  into  rabbits  accustomed  to  arsenic,  these  animals  manifested 
an  increase  of  white  corpuscles,  just  as  in  animals  injected  with  non- 
lethal  doses.  These  oscillations  in  the  number  of  leucocytes,  like 
those  which  have  been  observed  after  poisoning  by  trisulphide  of 
arsenic,  certainly  indicate  that  the  organism  and  its  defensive  cells 
behave  in  the  same  way  to  both  slightly  soluble  and  very  soluble  salts 
of  arsenic.  In  the  first  case  it  was  easy  to  demonstrate  that  the 
accumulation  of  leucocytes  in  the  blood  and  in  the  peritoneal  exu- 
dation terminated  in  the  ingestion  of  the  granules  of  trisulphide. 
With  potassium  arsenite,  it  was  not  so  easy  to  prove  the  point;  a 
chemical  analysis  of  the  elements  of  the  blood,  however,  has  given 
a  decisive  answer.  After  injecting  the  lethal  dose  of  this  soluble 
salt  into  rabbits  accustomed  to  arsenic,  Besredka  bled  them  in  order 
to  separate  the  plasma,  leucocytes  and  red  corpuscles.  Several 
experiments  made  on  these  rabbits  gave  a  concordant  result  which 
this  observer  sums  up  thus :  "  Although  the  bulk  of  plasma  and  of 
red  corpuscles  was  much  greater  than  that  of  the  leucocytes,  it  was 
in  the  latter  only  that  arsenic  was  found  "  by  chemical  analysis.  It 
was  only  in  those  cases  where  the  animals  survived,  and  manifested 
hyperleucocytosis,  that  Besredka  succeeded  in  demonstrating  the 
presence  of  arsenic  in  the  white  corpuscles. 

These  experiments,  excluding  any  doubt  as  to  the  protective  part 
played  by  the  leucocytes  against  arsenical  intoxication,  of  course 
suggested  the  idea  of  investigating  whether  the  nerve  elements,  sub- 
mitted to  the  direct  influence  of  potassium  arsenite,, exhibit  any  real 
susceptibility  to  this  poison.  The  injection  of  solutions  of  this  salt 
[419]  into  the  brain  demonstrated  that  the  one-hundredth  part  of  an  ordi- 
nary lethal  subcutaneous  dose  was  sufficient  to  cause  fatal  poisoning. 
This  fact,  then,  falls  into  line  with  other  facts,  already  numerous,  as 
to  the  susceptibility  of  the  nerve  centres  to  microbial  toxins,  alkaloids 


Artificial  immunity  against  toxins  399 

and  other  poisons.  But  in  the  case  of  potassium  arsenite,  it  was 
even  more  easily  demonstrated  than  in  the  other  cases  that  immunity 
natural  or  acquired,  is  connected  with  the  absorption  of  the  poison 
by  the  leucocytes.  These  cells,  themselves  much  less  susceptible  to 
the  toxic  action  than  are  the  nerve  elements,  protect  them  from 
contact  with  the  poison. 

It  is  manifest  that  arsenic  is  not  the  only  mineral  substance 
capable  of  being  absorbed  by  the  phagocytes,  and  there  are  already 
on  record  well  established  facts  in  support  of  this  thesis.  Some  time 
previous  to  the  researches  on  arsenical  poisoning  just  summarised, 
Robert,  then  in  Dorpat,  set  his  pupils,  Stender,  Samoiloff,  Lipsky 
and  others1  to  make  systematic  researches  on  the  fate  of  iron  in 
the  animal  organism.  For  this  purpose  these  observers  made  use 
of  a  very  soluble  preparation  of  iron — or  better  expressed,  as 
soluble  as  possible — Dr  Hornemann'syerrMm  oxydatum  saccharatum 
sohibile,  which  does  not  precipitate  in  alkaline  media.  They  proved 
that  a  small  quantity  of  the  iron  introduced  into  the  animal  is 
eliminated  by  the  kidneys  and  the  wall  of  the  intestine,  but  that 
the  greater  part  of  the  metal  is  arrested  in  the  organs,  especially 
the  liver,  spleen  and  bone  marrow.  The  iron  is  there  absorbed 
by  the  leucocytes  which  hold  it  for  some  time  and  then  throw  it 
into  the  intestine. 

I  have  had  the  opportunity  of  observing  this  circulation  of 
Dr  Hornemann's  soluble  salt  in  the  organism  of  several  species  of 
vertebrates.  Some  time  after  its  introduction  into  the  organism  by 
the  blood  vessels,  peritoueally  or  subcutaneously,  the  iron  may  be 
found  (by  means  of  the  microchemical  reaction  with  potassium 
ferrocyanide)  accumulated  in  the  various  phagocytes,  especially  the 
leucocytes,  the  stellate  Kupffer's  cells  of  the  liver  and  the  macro- 
phages  of  the  splenic  pulp.  The  non-phagocytic  cells,  as,  for  example, 
Ehrlich's  basophile  leucocytes,  so  abundant  in  the  lymph  of  rats, 
take  up  very  little  of  this  iron,  although  the  macrophages  and  micro- 
phages  are  full  of  it2.  Against  these  facts  Weigert3  has  advanced  [420] 
the  objection  that  the  leucocytes  absorb  only  the  iron  precipitated 
in  the  form  of  granules,  but  my  own  researches  allow  of  no  doubt 
that  not  only  granular  but  dissolved  iron  is  absorbed.  This  dis- 


1  Arl.  d.pharmak.  Instit.  z.  Dorpat,  1893,  1894,  Bde  vn— x. 
•  Ann.  de  VInst.  Pasteur,  Paris,  1894,  t  vm,  p.  719. 

3  Lubarsch    u.    Osier-tag's    Ergebnisse    d.    allg.    Path.,    Jahrg.    iv    for    189' 
Wiesbadeu,  1S99,  S.  107. 


400  Chapter  XII 

cussion,  however,  loses  much  of  its  importance  in  view  of  the  results 
obtained  with  potassium  arsenite. 

According  to  Samoiloff1,  soluble  salts  of  silver  in  the  animal 
organism  undergo  a  fate  similar  to  that  of  Hornemann's  soluble  iron 
salt  and  are  absorbed  by  the  phagocytic  elements.  It  must  be  noted, 
further,  that  according  to  the  experiments  of  Arnozan  and  Montel2, 
the  leucocytes  absorb  such  drugs  as  calomel  and  salicylate  of  soda. 

These  observations  all  clearly  show  that  the  phagocytes  must  not 
be  looked  upon  as  cells  capable  of  seizing  merely  the  dead  bodies  of 
micro-organisms  and  of  animal  cells,  always  fearing  and  avoiding 
poisons  and  only  able  to  come  forward  when  protected  by  some  other 
antitoxic  function.  The  phagocytes  no  doubt  often  exhibit  a  negative 
susceptibility  for  many  poisons,  when  these  are  introduced  into  the 
animal  organism  in  too  large  a  quantity.  But  these  cells  are  most 
resistant  to  toxic  substances  and  protect  the  higher  elements  from 
the  poison.  Under  these  conditions,  it  is  quite  natural  to  assign  to 
the  phagocytes  the  r61e  of  the  fighting  agents  of  the  animal  organism 
against  poisons  and  we  may  even  enquire  whether  these  elements  do 
not  produce  the  antitoxins.  It  has  been  pointed  out  that  it  is  very 
difficult  to  attribute  this  function  to  the  cells  susceptible  to  the  toxic 
action, — the  spermatozoa  in  the  production  of  antispermotoxin,  the 
red  blood  corpuscles  in  the  development  of  antihaemotoxin,  or  the 
nerve  cells  in  the  production  of  tetanus  antitoxin.  Moreover  since, 
according  to  Ehrlich's  theory,  it  is  only  the  haptophore  group  which 
excites  the  formation  of  antitoxins  on  the  part  of  the  elements  which 
possess  the  corresponding  receptors,  it  is  quite  possible  that  the 
phagocytes,  thanks  to  the  facility  with  which  they  absorb  the  poisons, 
occupy  an  important  place  as  producers  of  antitoxins.  I  have  already 
[421]  formulated  this  hypothesis,  and  several  investigators,  amongst  whom 
may  be  cited  Gautier3  and  Courmont4,  have  received  it  favourably, 
though  in  the  imperfect  state  of  our  knowledge,  it  cannot,  as  yet, 
be  fully  demonstrated.  It  might  perhaps  be  objected  against  this 
hypothesis  that  in  many  instances,  after  the  injection  of  micro- 
organisms living  or  dead,  in  spite  of  a  vigorous  leucocytic  reaction 
the  organism  of  the  animal  does  not  produce  any  antitoxin.  In  such 

1  Arb.  d.  pharmak.  Instil,  z.  Dorpat,  1893,  Bd.  ix,  S.  27. 

2  Communication  to  the  XHIth  Intern.  Congress  of  Medicine  in  Paris,  1900. 
"  Les  toxines  microbiennes  et  animales,"  Paris,  1896. 

4  In  Bouchard's  Traite  de  Pathologie  generate,  Paris,  1900,  t.  in,  2me  partie,. 
article  "  Inflammation." 


Artificial  immunity  against  toxins  401 

cases,  there  is  clearly  a  development  of  antibodies,  such  as  the 
fixatives,  whose  phagocytic  origin  may  reasonably  be  claimed,  but 
no  true  antitoxins.  It  must  not  be  forgotten,  however,  that  the 
various  kinds  of  phagocytes  present,  amongst  themselves,  great 
differences,  and  that  perhaps  certain  only  of  these  elements  are 
capable  of  producing  antitoxins.  When  micro-organisms,  living  or 
dead,  are  introduced  into  an  animal  it  is  found  that  antitoxins  do 
not  as  a  rule  appear  in  the  fluids;  in  these  cases  the  reaction  is 
set  up  mainly  by  the  microphages.  The  macrophages  represent 
the  principal  source  of  antitoxins.  In  cases  where  these  phagocytes 
ingest  the  micro-organism  the  blood  exhibits  an  undoubted  antitoxic 
power.  Such  is  the  case  with  bubonic  plague  in  the  human  subject, 
where  the  micro-organism  is  readily  ingested  by  the  macrophages. 
Here  we  obtain  antitoxic  serums  even  after  the  introduction  of  living 
or  dead  organisms  into  the  animal,  a  fact  observed  by  Roux  and 
his  collaborators.  Another  fact  in  favour  of  the  hypothesis  I  am 
defending  is  furnished  to  us  by  the  cayman.  As  noted  above,  this 
reptile,  of  all  known  animals,  supplies  antitoxins  most  quickly  and 
easily.  In  the  cayman  the  leucocytic  system  is  composed  of  eosino- 
phile  microphages  filled  with  granules,  and  of  macrophages.  As  the 
eosinophile  cells  are  only  very  weakly  phagocytic,  it  is  the  macro- 
phages almost  exclusively  which  intervene  in  the  reaction  against 
the  micro-organisms.  It  is  probable,  then,  that  in  the  cayman  and 
in  animals  inoculated  with  the  plague  bacillus  the  exclusion  of  the 
microphages  from  the  struggle  constitutes  a  factor  favourable  to  the 
production  of  antitoxins  and  at  the  same  time  favourable  to  the 
manifestation  of  the  activity  of  the  macrophages. 

If  these  latter  phagocytes  play  the  primary  role  in  the  excretion 
of  antitoxins  in  the  fluids  of  the  body  we  should  expect  to  find  this  [422] 
function  exercised  not  only  by  the  motile  macrophages  of  the  blood 
and  lymph,  but  also  by  the  fixed  macrophages,  so  widely  diffused 
through  almost  all  the  organs. 

I  advance  this  hypothesis  for  what  it  is  worth,  simply  as  a  guiding 
idea  for  new  researches  in  this  field,  of  which  so  much  is  still  un- 
known1. The  brief  account  of  the  actual  state  of  the  question  of 

1  Homer's  recent  researches  (Arch.  f.  Ophth.,  Leipzig,  1901,  Bd.  LIT,  S.  72)  on 
anti-abrin  accord  very  well  with  our  hypothesis.  He  was  able  to  demonstrate  that 
the  spleen,  the  bone-marrow,  and  the  conjunctiva  of  the  eye,  when  submitted  to  the 
influence  of  abrin,  contain  a  notable  quantity  of  anti-abrin.  Now  these  three  organs 
are  very  rich  in  phagocytes. 

B.  26 


402  Chapter  XII 

artificial  immunity  against  toxins,  has  indicated  to  us  that  this  is  a 
problem  far  more  difficult  of  solution  than  is  that  of  acquired  im- 
munity against  micro-organisms.  The  mere  fact  that  these  latter 
can  still  be  found  some  hours  or  even  days  after  their  entry  into 
the  refractory  animal,  affords  a  great  advantage  in  these  researches 
as  compared  with  those  on  toxins  which  are  lost,  often  almost  imme- 
diately, after  their  injection.  Consequently  our  knowledge  of  anti- 
microbial immunity  is  more  advanced  than  is  that  on  immunity  against 
the  soluble  products  of  micro-organisms. 

The  facts  narrated  in  this  chapter  support  the  thesis  I  have 
defended  on  the  subject  of  immunity  against  micro-organisms — that 
antimicrobial  immunity  in  no  way  depends  on  a  previous  resistance 
against  the  toxins.  As  a  general  rule  the  immunity  against  micro- 
organisms is  developed  more  readily  than  the  immunity  against 
their  toxic  products  and  at  an  earlier  stage. 

Although  much  still  remains  to  be  done  in  the  elucidation  of  the 
mechanism  of  antitoxic  immunity,  the  principal  data  acquired  on 
the  subject  of  this  immunity  have  undoubtedly  led  to  applications  of 
the  highest  importance,  as  will  be  set  forth  in  one  of  the  following 
chapters. 


CHAPTEE  XIII  [423] 

IMMUNITY  OF  THE  SKIN  AND  MUCOUS  MEMBRANES 

Protective  function  of  the  skin. — Exfoliation  of  the  epidermis  as  a  means  of  ridding 
the  animal  of  micro-organisms. — Localisation  and  arrest  of  micro-organisms  in 
the  dermis. — Intervention  of  phagocytes  in  the  defence  of  the  skin. 

Elimination  of  micro-organisms  by  the  conjunctiva.— Microbicidal  function  of  the 
tears. — Absorption  of  toxins  by  the  conjunctiva. — Protection  of  the  cornea. — 
Elimination  of  micro-organisms  by  the  nasal  mucosa. — Protection  by  the  respi- 
ratory channels. — Dust  cells. — Absorption  of  poisons  by  the  respiratory  channels. 

Alleged  microbicidal  property  of  the  saliva.— Part  played  by  microbial  products  in 
the  protection  of  the  buccal  cavity. — Antitoxic  function  of  the  saliva. 

Antiseptic  action  of  the  gastric  juice. — Antitoxic  function  of  pepsin. 

Protective  function  of  the  alimentary  canal. — Absence  of  microbicidal  power  from  the 
intestinal  ferments. — Protective  function  of  the  bile. — Antitoxic  r6le  of  the 
digestive  ferments. — Favouring  and  retarding  functions  of  the  intestinal  micro- 
organisms.— Destruction  of  toxins  by  these  micro-organisms. 

Defensive  role  of  the  liver.  Protective  function  of  the  lymphoid  organs  of  the 
alimentary  canal. 

Protective  function  of  the  mucous  membrane  of  the  genital  organs.— Autopurifica- 
tion  of  the  vagina. 

IN  the  preceding  chapters  the  phenomena  of  immunity  which  are 
exhibited  within  the  animal  body  in  which  the  portals  were  open  for 
the  penetration  of  the  micro-organisms  and  their  poisons  have  been 
studied.  We  had  to  deal  almost  exclusively  with  experimental  im- 
munity, the  study  of  which  constitutes  the  basis  of  our  present 
knowledge  concerning  the  general  problem  of  immunity.  In  natural 
immunity,  however,  things  do  not  follow  the  same  course.  The  micro- 
organisms and  their  toxins  are  not  introduced  directly  into  the  tissues 
and  blood  by  means  of  a  syringe  or  other  instrument.  The  micro- 
organisms have  to  make  their  own  way  through  the  skin  and  the 
mucosae,  tissues  which  offer  a  resistance  more  or  less  serious  and 

26—2 


404=  Chapter  XIII 

effective  ;  or  they  may  have  to  take  up  their  abode  in  the  cavities  of 
the  animal  organism,  in  order  that  they  may  be  able  to  inundate  it 
with  their  poisons.  We  must  here  review  briefly  these  natural  barriers 
to  microbial  invasion. 

The  skin  constitutes  a  protective  covering  of  great  importance  in 
[424]  connection  with  the  preservation  against  microbial  invasion  of  the 
delicate  parts  of  an  animal.  In  many  of  the  lower  and  higher  animals, 
and  even  in  man  himself,  the  skin  becomes  the  seat  of  a  microbial 
flora,  often  very  abundant,  in  which  may  be  found,  in  addition  to 
certain  inoffensive  organisms,  other  minute  parasites  more  or  less 
harmful.  The  pyogemc  cocci,  staphylococci  and  streptococci,  are 
constantly  found  on  the  human  skin,  most  frequently  hidden  in  the 
depths  of  the  canals  of  the  hair  follicles.  These  micro-organisms 
seize  every  favourable  opportunity  to  attack  the  organism,  producing 
such  local  lesions  of  the  skin  as  acne,  pimples,  boils,  and  erysipelas, 
or  even  becoming  generalised  in  the  blood  and  tissues,  as  in  the 
septicaemias  and  pyaemias.  To  the  skin,  therefore,  must  be  assigned 
a  very  important  function  in  the  prevention  of  the  invasion  of 
micro-organisms  which  are  found  constantly  on  the  surface  of  the 
body  or  which,  along  with  all  kinds  of  dirt,  are  brought  there 
accidentally. 

The  skin  is  able  to  fulfil  this  protective  function  from  the  fact 
that,  in  most  animals,  it  is  covered  with  a  not  very  permeable  layer 
of  some  considerable  thickness.  In  the  majority  of  the  Invertebrata, 
of  all  classes,  the  surface  of  the  body  is  clothed  with  a  chitinous  layer, 
sometimes  very  thin  and  capable  of  folding  and  following  all  the 
movements  of  the  body;  or  again  it  may  be  impregnated  with 
calcareous  salts  and  very  hard,  as  in  the  case  of  the  integument  of 
Insects  and  Crustacea,  and  the  shell  of  the  Mollusca.  In  all  cases 
this  cutaneous  sheath  constitutes  a  formidable  obstacle  to  the  entry 
of  micro-organisms.  Even  in  animals  of  very  small  size  the  thin 
cuticle  is  effective  in  preventing  any  invasion  by  these  parasites. 
Thus  the  Saprolegniae,  fungi  so  fatal  to  many  aquatic  animals,  are 
often  quite  unable  to  pass  through  this  cuticular  layer.  In  order  to 
pass  this  obstacle  their  germs  must  take  advantage  of  some  fissure 
or  wound,  produced  by  other  causes.  Daphniae,  too,  may  often  be 
observed  to  succeed  in  ridding  themselves  of  the  Monospora  with 
its  needle-like  spores  by  means  of  a  mechanism  which  we  have 
already  described  in  chapter  vi.  The  white  corpuscles  of  the  blood 
surround  the  spores  of  this  parasite  and  transform  them  into  an 


Immunity  of  the  skin  and  mucous  membranes    405 

innocuous  detritus.  Sometimes,  however,  a  number  of  these  fine 
spores  manage  to  perforate  the  cutaneous  investment  of  the  small 
crustacean ;  quite  a  small  opening  is  made  in  the  chitinous  wall, 
which  in  itself  is  a  source  of  no  danger.  As  soon,  however,  as  a  spore 
of  the  Saprolegnia  approaches  this  opening,  it  immediately  begins  to 
thrust  a  process  through  the  small  lesion,  and  from  that  moment  the 
fate  of  the  Daphnia  is  sealed.  Incapable  of  opposing  the  slightest  [425] 
phagocytic  resistance  to  the  filaments  of  the  fungus,  it  is  invaded 
throughout  by  the  mycelium  and  soon  dies. 

The  integrity  of  the  skin  being  so  important  for  the  preservation 
of  life,  a  fairly  perfect  mechanism  has  been  elaborated  for  the 
maintenance  of  this  integrity.  All  animals,  no  matter  what  their 
position  in  the  animal  scale,  are  liable  to  lesions  and  wounds  of  the 
surface  of  their  bodies.  In  the  Daphniae  I  have  often1  observed 
wounds  produced  by  the  bites  of  other  aquatic  animals.  The  surface 
of  these  wounds  soon  becomes  covered  with  a  rich  microbial  vegeta- 
tion. The  leucocytes  are  brought  up  to  the  injured  point  and  there 
produce  a  protective  layer ;  but,  at  the  same  time,  a  rapid  proliferation 
of  the  neighbouring  cells  of  the  epidermis  takes  place;  this  closes  the 
wound  and  separates  the  skin,  so  reconstituted,  from  the  micro- 
organisms. Everything  resumes  its  original  order  and  the  leucocytes 
soon  disperse,  regaining  the  blood  stream. 

These  phenomena,  which  can  be  readily  observed  under  the 
microscope  in  such  small  and  transparent  animals  as  the  Daphniae, 
may  serve  as  the  prototype  of  those  of  a  number  of  analogous 
processes  in  the  animal  kingdom.  The  thicker  and  more  solid  the 
cuticular  investment,  the  more  fully  it  guarantees  the  animal  against 
the  penetration  of  micro-organisms.  Cue*not2  made  the  observation 
that  Crustacea,  furnished  with  such  a  hard  envelope  as  is  the  carapace 
of  the  Decapods,  are  completely  defenceless  from  the  moment  parasitic 
micro-organisms  make  their  way  into  their  bodies.  These  intruders 
quietly  instal  themselves  in  the  tissues,  without  causing  the  slightest 
phagocytic  reaction,  and  thus  bring  about  the  inevitable  death  of  the 
host.  The  protection  of  the  animal  in  this  case  is,  so  to  speak, 
associated  with  the  resistance  offered  by  the  carapace. 

Again,  in  many  of  the  Vertebrata,  the  skin  has  a  hard,  thick 
sheath,  e.g.,  the  scales  of  fishes  and  of  reptiles.  Man,  with  his 
supple  and  not  very  thick  skin,  is  less  well  endowed ;  this,  however, 

1  Vir chow's  Archie,  Berlin,  1884,  Bd.  xcvi,  S.  192. 
"  Arch,  de  Biol^  Gand  et  Leipzig,  1893,  t.  xm,  p.  245. 


406  Chapter  XIII 

does  not  prevent  him  from  defending  himself  against  the  entry  of 
micro-organisms  by  the  cutaneous  path.  Sabouraud1,  a  well-known 
dermatologist,  has  given  a  very  concise  and  at  the  same  time  very 
complete  sketch  of  the  part  played  by  the  skin  in  the  protection 
[426]  of  the  body  against  micro-organisms  ;  from  this  author  the  following 
data  are  borrowed. 

The  epidermic  layer  sets  up  a  defence  by  the  production  and 
expulsion  of  corneal  cells.  In  the  normal  course  of  the  life  of  the 
epidermis,  the  cells  of  the  deeper  layers,  coming  to  the  surface,  be- 
come exfoliated  and  are  thrown  off.  "There  is  thus  produced,  a 
continual  exfoliation  of  the  dead  layers,  and  a  continual  eviction 
of  such  micro-organisms  as  are  living  on  them.  The  epidermis  is 
dense  and  its  cells  have  a  hard  envelope ;  the  micro-organism  is 
not  endowed  with  motion,  or  at  least  not  with  sufficient  to  be  of 
service  in  effecting  an  entrance.  It  can  only  penetrate  the  epidermis 
by  multiplication  in  situ,  a  micro-organism  originates  alongside 
another,  another  in  front  of  it,  and  in  front  of  this  again  others.  In 
this  way  they  burrow  between  the  apposed  cells  just  as  a  root 
penetrates  into  the  ground ;  so  great  is  the  resistance  of  the  horny 
cells  that  we  never  find  any  micro-organisms  within  them,  but 
between  them  only  "  (p.  734).  The  epidermic  cells,  containing  micro- 
organisms, exfoliate,  and  the  skin  is  thus  ridded  of  them.  Frequently 
the  process,  as  it  goes  on  constantly  and  slowly,  is  invisible ;  but 
often,  on  the  other  hand,  it  becomes  exaggerated  and  manifests  itself 
in  the  form  of  a  cuticular  desquamation  which  leads  to  the  elimination 
of  a  large  number  of  micro-organisms.  The  patient  may  retain  "  such 
pellicles  for  ten  years,  and  even  longer,  without  presenting  anything 
else  but  these,  and  there  are  many  other  chronic  squamous  infections 
in  which  the  course  is  uncomplicated  by  even  an  erosion  or  the 
slightest  wound." 

The  connective  tissue  of  the  human  skin  is  also  fully  able  to 
defend  itself ;  it  is  extremely  vigorous  and  represents  a  real  obstruct- 
ing and  resisting  tissue.  The  penetration  of  parasites  sets  up  in  it 
a  thickening  of  the  fibrous  tissue ;  this  effects  a  localisation  of  the 
microbial  focus.  To  appreciate  the  effectiveness  of  this  dermic  de- 
fence, we  have  only  to  compare  the  slow  growth  of  lupus,  a  form  of 
cutaneous  tuberculosis,  with  that  of  tuberculosis  of  the  lungs  and 
other  viscera,  or  the  slow  evolution  of  farcy,  or  cutaneous  glanders, 
with  that  of  the  visceral  form  of  the  disease. 

1  Ann.  de  dertnat.  et  de  syph.,  Paris,  1900,  t.  x,  p.  729. 


Immunity  of  the  skin  and  mucous  membranes    407 

If  we  examine  more  closely  the  process  by  which  the  dermis  sur- 
rounds the  intruders  with  a  fibrous  capsule,  we  readily  recognise  in  it 
a  reaction  of  the  macrophages  of  the  skin.  In  lupus  these  phagocytes 
seize  the  tubercle  bacilli,  combining  to  form  giant  cells  and  giving 
rise  to  an  exaggerated  development  of  the  connective  tissue  fibres. 
Moreover,  when  the  skin  is  menaced  with  a  microbial  invasion,  not 
only  the  local  macrophages  but  the  leucocytes  are  mobilised.  The  [427] 
migratory  white  corpuscles  travel  through  the  epidermis  and  the 
connective  tissue  layer.  In  spite  of  the  absence  of  a  lymphatic 
circulation  in  the  epidermis,  the  leucocytes  penetrate  into  this  layer 
"  and,  in  a  section  through  the  normal  epidermis,  it  is  very  rare  not  to 
find  here  and  there  some  deformed  and  flattened  leucocyte,  surprised 
just  as  it  was  creeping  between  the  cells  of  the  rete  mucosa  or  of  the 
stratum  granulosum"  Immediately  that  the  epidermis  or  the  dermis 
finds  itself  menaced  with  a  microbial  invasion,  an  accumulation  of 
leucocytes  of  all  kinds  is  produced  at  once ;  this  may  remain 
microscopic  or  it  may  assume  proportions  visible  to  the  naked  eye. 
Frequently  the  subjacent  epithelium  throws  off  epidermic  scales 
which  are  filled  with  leucocytes ;  often  also  the  leucocytic  foci  in 
the  dermis  become  emptied,  the  micro-organisms  being  expelled 
along  with  their  enemies  the  phagocytes. 

The  tissues  of  the  skin  proper  defend  themselves  against  micro- 
organisms as  well  as  they  are  able ;  but  so  soon  as  the  danger 
becomes  serious  there  is  sent  to  their  succour  a  whole  army  of  mobile 
phagocytes.  This  example  of  the  defence  made  by  the  cutaneous 
investment  may  serve  as  a  prototype  of  that  of  every  other  region 
of  the  body.  Alongside  a  local  action,  there  is  always  an  intervention 
of  mobile  phagocytes ;  but  when  this  action  becomes  insufficient, 
a  much  more  abundant  accumulation  of  leucocytes  than  is  found 
in  ordinary  cases  is  immediately  produced. 

Like  the  skin,  the  mucous  membranes  are  invested  with  an 
epithelial  layer,  which  serves  as  a  barrier  to  the  entry  of  micro- 
organisms. But  whilst  the  surface  of  the  normal  skin  is  dry  or  barely 
moistened  by  the  secretory  products  of  the  cutaneous  glands,  the 
mucous  membranes  are  always  humid,  a  condition  favourable  to  the 
multiplication  of  micro-organisms.  Hence  the  mucous  membranes 
which  are  most  exposed  to  contact  with  the  air  and  with  external 
objects,  always  contain  a  larger  or  smaller  number  of  organisms, 
amongst  which  the  pathogenic  species,  notably  staphylococci,  pneumo- 
cocci  and  streptococci,  are  the  most  common.  The  part  played  by 


408  Chapter  XIII 

the  animal  organism  in  getting  rid  of  these  micro-organisms  becomes 
more  complicated  than  in  the  case  of  the  defence  made  by  the  skin 

The  first  of  the  mucous  membranes  to  be  exposed  to  contamination 
by  micro-organisms  is  the  conjunctiva  of  the  eye.  At  the  moment 
of  birth  it  is  in  contact  with  the  vaginal  mucous  membrane  and 
acquires  from  it  some  of  its  micro-organisms,  botli  innocuous  and 
pathogenic.  Tears  fulfil  the  function  of  averting  the  danger  resulting 
[428]  from  this  proximity  and  from  the  presence  of  micro-organisms  in  the 
conjunctival  sac  in  general.  Ophthalmologists  have  shown  that  these 
tears  transport  the  organisms  into  the  nasal  cavity  by  means  of  the 
lachrymal  canal.  To  determine  this  point  Bach1  introduced  a  number 
of  Kiel  water  bacilli  along  with  pyogenic  staphylococci  into  the  con- 
junctival sac  of  various  individuals.  Seedings  made  with  the  tears 
showed  a  very  rapid  disappearance  of  the  two  organisms,  which  passed 
into  the  nose  where  their  presence  could  be  demonstrated  by  making 
plate  cultures  of  the  nasal  mucus.  Enormous  numbers  of  the  Kiel 
bacilli,  introduced  into  the  conjunctival  sac,  were  all  transferred  to 
the  nasal  cavity,  on  the  average,  by  the  end  of  half-an-hour.  The 
pyogenic  staphylococci  persisted  on  the  surface  of  the  conjunctiva 
for  a  longer  period,  but  they  also  passed  in  large  numbers  through 
the  lachrymal  canal  into  the  nose. 

Certain  observers,  notably  Bernheim2,  thought  that  the  tears, 
in  addition  to  their  purely  mechanical  defensive  action,  were  capable 
of  destroying  the  micro-organisms  by  their  microbicidal  power. 
Bach3  submitted  this  question  to  a  minute  examination  and  came  to 
the  conclusion  that  several  species  of  bacteria,  introduced  in  vitro 
into  the  tears  of  healthy  persons  or  of  those  who  were  suffering 
from  conjunctivitis  or  certain  other  ocular  diseases,  disappeared 
somewhat  rapidly.  Comparative  experiments  with  tears  previously 
heated  to  58°  and  even  to  70°  C.,  in  most  cases  gave  the  same 
results,  that  is  to  say,  they  caused  a  rapid  disappearance  of  the 
organisms  introduced.  From  these  facts  the  author  concluded  that 
it  is  probably  to  the  salts  contained  in  the  tears  that  their  bacteri- 
cidal action  is  due.  Control  experiments  made  with  physiological 
saline  solution  and  with  various  mixtures  of  mineral  salts  met 
with  in  the  tears  have  been  found  by  Bach  to  cause  a  like 
disappearance  of  the  same  species  of  organisms.  Well  water,  and 

1  von  Graefe's  Arch.  f.  Ophth.,  Leipzig,  1894,  Bd.  XL,  S.  130. 

2  Deutschmann's  Beitr.  z.  Augenheilk,  Hamburg  m  Leipzig,  1893,  Hft.  VIII. 
8  op.  cit.  supra. 


Immunity  of  the  sJcin  and  mucous  membranes    409 

even  distilled  water,  gave  the  same  result.  In  all  these  cases  it  is 
evident  that,  in  the  tears,  there  is  no  bactericidal  cytase  comparable 
with  that  found  in  the  serums  and  other  body  fluids  which  may 
contain  this  phagocytic  diastase.  The  experiments  with  heated  tear& 
demonstrate  this  clearly.  On  the  other  hand,  these  same  experiments 
lead  one  to  suppose  that  the  diminution  and  even  the  disappearance  [429] 
of  the  micro-organisms  in  the  tears,  is  due  to  a  large  extent,  and 
perhaps  completely,  to  an  agglutinative  action  of  the  salts,  a  fact 
which  has  been  demonstrated  by  several  observers. 

In  all  these  cases  it  is  indisputable  that  the  mechanical  part 
played  by  the  tears  is  the  most  important  of  the  defences  offered 
by  the  conjunctiva  of  the  eye  against  the  microbial  invasion.  That 
this  defence  is  not  always  sufficient  is  proved  by  the  frequency  of 
conjunctivitis,  as  well  as  by  the  ease  with  which  certain  micro- 
organisms, inoculated  into  the  conjunctival  sac,  set  up  a  general 
infection.  This  is  specially  the  case  with  the  coccobacillus  of 
human  plague.  When  it  is  introduced  into  the  conjunctival  sac 
of  susceptible  animals  (rat,  guinea-pig,  &c.),  it  passes  thence  into 
the  nasal  cavity  and  soon  produces  a  generalised  and  fatal  infection. 
The  conjunctival  membrane,  even  when  perfectly  intact,  readily 
absorbs  certain  poisons.  Everyone  knows  the  rapidity  with  which 
atropin,  when  introduced  into  the  conjunctival  sac,  causes  dilatation 
of  the  pupil.  But  the  mucous  membrane  may  serve  also  as  the 
port  of  entry  for  toxins  of  microbial  origin.  Several  observers, 
and  especially  Morax  and  Elmassian1,  have  demonstrated  that  the 
diphtheria  poison  placed  upon  an  unbroken  conjunctival  membrane, 
where  the  epithelial  layer  is  uninjured,  sets  up  local  lesions  which 
progress  very  slowly  but  which  terminate  in  the  formation  of  actual 
false  membranes.  Nevertheless,  it  must  be  admitted  that  the  intact 
epithelial  layer  of  the  conjunctiva  exerts  a  certain  defensive  action 
against  the  penetration  of  toxins,  although  a  very  slight  lesion  of  this 
layer  will  allow  of  the  ready  absorption  of  the  diphtheria  poison  and 
the  formation  of  false  membranes. 

The  cornea  likewise,  so  long  as  it  is  intact,  exhibits  a  marked 
resistance  against  the  penetration  of  micro-organisms  and  of  toxins. 
When  it  becomes  injured  in  any  way  its  epithelium  is  repaired  with 
great  rapidity,  as  has  been  well  demonstrated  by  Ranvier2,  who  has 
shown  that  the  walls  of  the  wound  close  by  a  process  of  epithelial 

1  Ann.  de  tlnst.  Pasteur,  Paris,  1898,  t.  xii,  p.  210. 

2  Arch,  d'anat.  microsc.,  Paris,  1898,  t.  n,  pp.  44,  177. 


410  Chapter  XIII 

"  soldering  "  in  a  purely  mechanical  fashion,  without  the  intervention 
of  any  preliminary  proliferation  of  the  epithelial  elements.  Thanks 
to  this  very  rapid  obliteration  the  micro-organisms  are  prevented 
from  penetrating  not  only  into  the  interior  of  the  cornea,  but  into  the 
anterior  chamber  of  the  eye. 

[430]  It  has  already  been  pointed  out  that  the  ocular  conjunctiva  gets 
rid  of  the  introduced  micro-organisms  chiefly  by  removing  them 
mechanically  and  sending  them  through  the  lachrymal  duct  into  the 
nasal  cavity.  This,  in  turn,  defends  itself  by  making  use  of  a  similar 
method.  In  his  experiments  on  the  Kiel  red  bacillus,  inoculated  into 
the  conjunctival  sac  of  man,  Bach  demonstrated  that  in  a  very  short 
time  these  micro-organisms  are  carried  into  the  nasal  cavity.  He 
showed  also  that  they  do  not  remain  long  in  the  latter  position  and 
that  their  number  decreases  hourly. 

Twenty-four  hours  after  the  introduction  of  these  bacilli  into  the 
conjunctiva  none,  as  a  general  rule,  are  to  be  found  in  the  nasal 
mucus.  This  expulsion  of  the  micro-organisms  likewise  takes  place 
by  mechanical  means,  aided  by  the  movements  of  the  vibratile  cilia. 
It  is  evidently  to  this  process  that  the  mucous  membrane  owes  its 
relative  freedom  from  micro-organisms.  Frequently,  when  examining 
the  nasal  mucus  or  when  making  cultures  therefrom,  one  is  astonished 
at  the  small  number  of  micro-organisms  found  in  the  nasal  cavities 
of  persons  in  good  health.  Thomson  and  Hewlett1  have  certainly  gone 
too  far  when  they  affirm  that  the  upper  regions  [i.e.  the  Schneiderian 
membrane]  of  the  nasal  cavity  are,  in  almost  80  °/0  of  cases,  free  from 
micro-organisms.  But  it  is  certain  that  in  these  regions  we  do  find  a 
small  number  only  of  the  bacteria  which  exist  in  greater  abundance 
in  the  lower  (cutaneous)  passages  of  the  nose. 

To  explain  this  paucity  of  micro-organisms  in  the  nasal  cavity, 
Wurtz  and  Lermoyez2  have  assumed  the  existence  of  a  bactericidal 
property  in  the  nasal  mucus.  They  affirm  that  the  anthrax  bacillus, 
after  contact  with  this  mucus  for  several  hours,  loses  its  virulence  for 
the  most  susceptible  animals,  and  that  several  other  micro-organisms 
—the  staphylococci,  the  streptococci,  and  the  Bacillus  coli— become 
attenuated  under  the  same  conditions.  Others  who  have  studied 
this  question  have  come  to  a  different  conclusion.  Thomson  and 
Hewlett  found  that  the  nasal  mucus  is  not  bactericidal,  although 

1  [Med.-Chir.  Trans.,  London,  1895,  Vol.  LXXVIII,  p.  239];  The  Lancet,  London, 
1896,  Vol.  i,  p.  86 ;  Brit.  Med.  Journ.,  London,  1896,  Vol.  I,  p.  137. 

2  Compt.  rend.  Soc.  de  biol,  Paris,  1893,  p.  756. 


Immunity  of  the  skin  and  mucous  membranes    411 

it  prevents  the  multiplication  of  micro-organisms.  F.  Klemperer, 
denies  the  bactericidal  property  of  the  nasal  mucus.  He  could  never 
demonstrate  the  destruction  of  micro-organisms  by  the  mucus,  and 
he  also  observed  that  bacteria  do  not  multiply  at  all  readily  in  this 
medium.  These  results  confirm  the  hypothesis  that  the  defensive  [431] 
action  of  the  nasal  mucous  membrane  against  microbial  invasion 
is  mainly  effected  by  the  mechanical  elimination  of  the  numerous 
micro-organisms  which  continually  reach  it.  Amongst  these  orga- 
nisms are  some  which  are  conspicuous  for  the  ease  with  which 
they  multiply  in  the  body,  taking  the  nasal  cavity  as  a  starting  point, 
e.g.  the  micro-organisms  of  influenza,  the  plague  bacillus,  which, 
according  to  several  observers,  is  very  virulent  when  introduced  by 
the  nostrils2,  and  the  leprosy  bacillus.  This  last,  according  to 
Goldschinidt3,  Sticker4,  and  Jeanselme5  often  enters  the  human  body 
by  way  of  the  nose. 

It  is  certain  that  the  olfactory  apparatus  deprives  the  inspired  air 
of  a  large  number  of  the  micro-organisms  which  it  carries.  These 
organisms  deposited  on  the  mucous  membrane  are  expelled  with  the 
nasal  mucus.  A  number  of  the  foreign  organisms,  carried  by  the  air, 
may,  however,  surmount  this  first  barrier  and  penetrate  further  into 
the  trachea  and  bronchi,  whence,  helped  by  the  movements  of  the 
vibratile  cilia,  they  are  usually  expelled  along  with  the  mucus. 

In  spite  of  this  double  defence  it  has  been  recognised  that  very 
minute  corpuscles  and,  amongst  others,  micro-organisms  may  over- 
come every  one  of  these  obstacles  and  reach  the  pulmonary  alveoli. 
Here,  under  the  name  of  "dust-cells"  ("cellules  &  poussiere ")— 
"  Staubzellen  "  of  the  German  writers — located  in  the  alveoli,  are  de- 
scribed certain  large  mouonucleated  elements  which  contain  granules 
of  foreign  origin,  usually  deposits  of  soot,  of  a  deep  black.  This 
permeability  of  the  normal  lung  tissue  for  dust  particles  and  pig- 
mented  corpuscles  has  been  closely  studied  and  clearly  demonstrated 
by  J.  Arnold6  and  his  pupils.  Several  observers  have  tried  to 
determine  whether  micro-organisms,  introduced  by  the  respiratory 

1  Munchen.  med.  Wchnschr.,  1896,  S.  730. 

2  Batzaroff,  "La  pneumonie  pesteuse  experimentale,"  Ann.  de  Flnst.  Pasteur* 
Paris,  1899,  t.  xm,  p.  385. 

3  "La  Lepre,"  Paris,  1894. 

4  Munchen.  med.  Wchnschr.,  1897,  S.  1063. 
6  Presse  med.,  Paris,  1899,  8  avril. 

Untersuchungen  uber  Staubinhalation,"  Leipzig,  1885. 


6    « 


412  Chapter  XIII 

channels,  behave  like  other  bodies.  Animals  were  made  to  inhale, 
or  there  were  introduced  into  the  trachea,  cultures  of  bacteria 

[432]  pathogenic  for  the  animals  experimented  upon.  The  results  so  ob- 
tained have  been  very  contradictory.  Morse1,  Wyssoko witch2,  and 
Hildebrandt3,  never  succeeded  in  inducing  anthrax  by  the  intro- 
duction of  anthrax  bacilli  into  the  lungs  of  normal  animals.  They 
concluded,  therefore,  that  the  uninjured  pulmonary  tissue  was 
impermeable  by  virulent  micro-organisms.  H.  Buchner*  with  his 
collaborators  and  pupils  maintaining  the  opposite  view,  declare  that 
rabbits  that  have  inhaled  anthrax  bacilli  or  their  spores  always  suc- 
cumb to  a  fatal  attack  of  anthrax.  These  contradictory  results  were 
attributed  to  differences  in  the  methods  employed,  and  an  attempt 
was  made  to  perfect  the  methods  of  research,  especially  to  prevent 
the  penetration  of  the  anthrax  bacilli  by  lesions  of  the  trachea 
or  by  any  channel  other  than  that  of  the  pulmonary  tissue. 
Gramatschikoff5,  under  Baumgarten's  direction,  undertook  a  series 
of  experiments  in  order  to  determine  whether  it  was  possible  for  the 
anthrax  bacillus  to  traverse  the  pulmonary  tissue.  He  introduced 
through  the  trachea  of  rabbits  and  guinea-pigs  an  anthrax  culture, 
afterwards  washing  the  respiratory  passages  with  a  large  quantity  of 
broth  or  of  physiological  saline  solution.  Several  of  the  animals  so 
treated  did  not  succumb  to  the  inoculation,  and  Gramatschikoff  con- 
cluded that  it  was  impossible  for  the  anthrax  bacillus  to  make  its  way 
through  the  wall  of  the  normal  pulmonary  tissue.  He  was  satisfied 
that  some  of  the  injected  organisms  were  destroyed  in  the  lung, 
although  he  was  unable  to  see  how  this  bactericidal  action  was 
determined.  In  these  experiments  a  large  quantity  of  fluid  was  intro- 
duced after  the  bacilli ;  this  might  wash  away  the  bacilli  and  convey 
them  to  situations  where  they  could  exert  no  morbific  action; 
moreover  the  anthrax  bacilli  used  were  of  doubtful  virulence  (the 
injections  made  to  control  the  virulence  in  the  subcutaneous  tissue 
were  in  nearly  every  instance  made  with  quantities  of  fluid  greater 
than  those  introduced  by  the  trachea),  and  Gramatschikoff's  results 

[433]  could  not  be  accepted  as  deciding  the  question.    On  the  other  hand, 

"  Eingangspforten  der  Infectionsorganismen,"  Berlin,  1881. 
2  M itth.  aus  der  Brehmer'schen  Heilanstalt,  1899,  S.  297. 

"  Experim.  Unters.  ii.  d.  Eindringen  path.  Microorganismen,"  Konigsberg,  1888, 
[and  in  Ziegler's  Beitr.  z.  path.  Anat.,  Jena,  1888,  Bd.  n,  S.  411]. 

Arch.f.  Hyg.,  Munchen  u.  Leipzig,  1887,  Bd.  vm,  S.  145. 

'  Baumgarten's  Arb.  auf  d.  Geb.  d.  path.  Anat.  etc.,  Braunschweig,  1892,  Bd.  i, 
S.  450. 


Immunity  of  the  skin  and  mucous  membranes    413 

H.  Buclmer's  inhalation  experiments  made  with  spores,  and  the 
study  of  the  organs  of  animals  so  treated,  leave  no  doubt  as  to  the 
possibility  of  the  invasion  of  an  animal  by  the  respiratory  channels 
by  the  anthrax  bacillus.  Furthermore,  the  "rag-picker's  disease" 
and  the  "  wool-sorter's  disease,"  or  pulmonary  anthrax,  developed  in 
man  as  a  result  of  the  inhalation  of  dust  charged  with  anthrax  spores, 
demonstrate  most  clearly  that  it  is  possible  for  the  anthrax  bacillus 
to  enter  the  body  by  the  respiratory  channels.  The  pulmonary 
mycoses,  produced  by  the  penetration  of  the  Aspergillus  fumigatus 
in  the  human  subject,  offer  confirmatory  evidence. 

In  spite  of  the  fact  that  the  pulmonary  tissue  is  not  impermeable 
to  pathogenic  micro-organisms,  it  is  none  the  less  true  that  it  exhibits 
a  very  marked  resistance  to  infection  by  this  channel.  It  is,  however, 
neither  the  thickness  of  the  wall,  as  in  the  case  of  the  skin  and  the 
mucous  membranes,  nor  the  mechanical  elimination  with  the  help  of 
the  vibratile  cilia  or  of  the  secretions,  that  constitute  the  means 
of  defence  in  the  respiratory  alveoli.  Here  the  cell  elements  are 
charged  with  the  duty  of  ridding  the  lungs  as  much  as  possible 
of  the  micro-organisms  which  enter.  Ribbert1  and  his  Bonn  pupils, 
Fleck2  and  Laehr3,  observed  this  fact  long  ago.  They  showed  that 
the  spores  of  Aspergillus  flavescens  and  the  staphylococci,  injected 
into  the  veins  or  into  the  trachea,  penetrate  into  the  pulmonary 
alveoli,  where  they  are  soon  seized  by  the  "  epithelial  cells  "  and  the 
leucocytes.  Laehr  observed  that  this  phenomenon  is  produced  at  the 
end  of  a  few  hours,  and  that  the  ingested  cocci  within  the  phagocytes 
undergo  a  progressive  degeneration  and  at  last  disappear.  Tchisto- 
vitch4,  working  in  my  laboratory,  studied  micro-organisms  pathogenic 
for  the  rabbit — the  anthrax  bacillus,  the  coccobacillus  of  fowl  cholera, 
and  the  bacillus  of  swine  erysipelas — ingested  by  the  "dust-cells"  of 
the  alveoli.  He  has  added  the  important  observation  (already  referred 
to  in  chapter  iv)  that  these  phagocytic  elements  are  not  epithelial  cells  [434] 
at  all,  but  are  really  macrophages  of  lymphatic  origin.  They  are  not 
found  in  the  alveoli  of  new-born  animals,  but  soon  appear  there  and 
iustal  themselves  in  such  a  manner  that  for  long  one  was  led  to 
regard  them  as  true  epithelial  cells  of  the  pulmonary  tissue.  This 
tissue,  invested  with  an  extremely  delicate  covering,  is  incapable  of 

1  "Dor  Untergang  pathog.  Schimmelpilze  im  Korper,"  Bonn,  1887. 

2  "  Die  acute  Entziindung  der  Lunge,"  Bonn,  1886. 

3  "  Ueb.  d.  Untergang  des  Staphylococcus,"  etc.,  Bonn,  1887. 

4  Ann.  de  I'Inst.  Pasteur,  Paris,  1889,  t.  m,  p.  337. 


414  Chapter  XIII 

defending  itself  against  the  invasion  of  micro-organisms,  but  the 
animal  organism  comes  to  its  aid  by  sending  a  permanent  army  of 
macrophages  which  evict  from  the  alveoli,  so  far  as  is  possible,  both 
micro-organisms  and  other  foreign  bodies.  Under  these  conditions, 
we  can  readily  understand  that  similar  cells  which  fulfil  the  same 
protective  function,  are  also  found  in  the  neighbouring  bronchial 
glands.  It  has  long  been  recognised  that  the  macrophages  of 
these  glands  are  often  crammed  with  various  kinds  of  granules 
of  foreign  origin,  which  have  made  their  way  into  the  lungs  with  the 
inspired  air. 

Toxic  substances  can  be  absorbed  by  the  mucous  membrane  of 
the  respiratory  channels.  Roger  and  Bayeux1  have  shown  that  no 
lesion  is  required  in  order  that  diphtheria  poison  may  invade  the 
mucous  membrane  of  the  trachea,  and  so  produce  typical  false  mem- 
branes. The  lung,  we  know,  is  accessible  to  gaseous  toxic  substances ; 
moreover,  its  surface  readily  absorbs  fluid  poisons. 

The  protection  of  the  digestive  system  is  more  complex  than 
that  of  the  respiratory  passages ;  this  is  not  remarkable,  when  we 
consider  the  greater  complexity  of  the  organs  of  digestion  and 
the  varied  conditions  which  they  present  with  regard  to  microbial 
invasion. 

The  buccal  cavity,  so  exposed  to  the  entry  of  extraneous  micro- 
organisms along  with  the  food  and  the  external  air,  has  a  very  rich 
microbial  flora,  in  which  Miller2,  the  author  of  our  most  complete 
work  on  this  subject,  has  recognised  in  man  more  than  thirty  species. 
Several  representatives  of  this  flora,  e.g.  the  Leptothrix  and  the 
Spirochaeta  are  constantly  present,  and  are  very  characteristic  of 
the  buccal  cavity  of  man.  With  them  are  frequently  found  pneumo- 
[435]  cocci,  staphylococci,  and  streptococci,  whose  pathogenic  power  is 
undoubted.  Virulent  diphtheria  bacilli  are  also  met  with  in  a  certain 
number  of  quite  healthy  persons.  It  is  astonishing  that,  in  spite  of 
this  state  of  things,  wounds  in  the  mouth  heal  very  rapidly,  and 
operations  on  the  buccal  cavity  done  with  insufficient  or  no  aseptic 
precaution  do  not,  in  the  great  majority  of  cases,  set  up  infective 
complications  of  the  slightest  importance.  After  certain  buccal 
operations  we  are  often  confronted  with  a  complicated  and  open 
fissure ;  nevertheless  the  wound  thus  left  exposed  is  not  ordinarily 
the  seat  of  any  infection  either  local  or  generalised. 

1  Compt.  rend.  Soc.  de  biol.,  Paris,  1897,  p.  265. 

2  "  Die  Mikroorganismen  der  Mundhohle,"  Leipzig,  2te  Aufl.,  1892. 


Immunity  of  the  skin  and  mucous  membranes    415 

It  is  often  asked,  how  under  these  conditions  does  the  mouth 
defend  itself  against  the  vast  number  of  formidable  micro-organisms. 
When  the  theory  of  the  bactericidal  power  of  the  body  fluids  was 
dominant,  and  appeared  to  explain  several  important  points  in  the 
general  problem  of  immunity,  the  saliva  was  studied  from  this 
"bactericidal"  point  of  view.  Sanarelli1,  as  the  outcome  of  patient 
and  laborious  researches,  came  to  the  conclusion  that  the  human 
saliva  acted  as  an  antiseptic  and  destroyed  a  large  number  of  micro- 
organisms. It  is  true  that  he  recognised  its  efficacy  only  when  few 
bacteria  were  subjected  to  its  action ;  but  even  when  the  saliva  was 
incapable  of  killing  a  large  number  of  micro-organisms,  it  did  not 
allow  them  to  develop — it  was  a  bad  culture  medium ;  moreover,  it 
had  the  power  of  attenuating  the  virulence  of  certain  pathogenic 
bacteria,  such  as  the  pneumococcus,  so  frequently  found  in  the 
mouth. 

The  conclusions  of  the  Italian  observer  did  not,  however,  meet 
with  general  acceptance.  Miller2  did  not  believe  that  the  saliva 
exerted  any  bactericidal  action,  raising  the  objection  that  the  absence 
of  nutritive  value  in  the  human  saliva  for  bacteria  is  explained  by 
the  fact  that  in  his  experiments  Sanarelli  employed  filtered  saliva, 
which  consequently  had  been  deprived  of  much  of  its  nutritive 
substances, — epithelial  debris,  mucus,  etc.  Hugenschmidt3,  working 
in  my  laboratory,  carried  out  a  special  research  on  the  influence  of 
the  human  saliva  on  micro-organisms,  and  arrived  at  conclusions 
quite  at  variance  with  those  reached  by  Sanarelli.  In  spite  of  the 
variety  of  micro-organisms  made  use  of,  he  could  never  satisfy  himself 
that  the  saliva  had  any  bactericidal  property. 

He  sometimes  saw,  no  doubt,  a  certain  slowness  of  growth  or  [436] 
even  the  destruction  of  certain  of  the  micro-organisms  sown  at  the 
commencement  of  the  experiment,  but  this  was  very  slight  and 
rather  exceptional.  In  most  cases  the  micro-organisms,  introduced 
into  the  saliva,  grew  rapidly,  so  that  their  number,  in  a  very  short 
time,  became  very  considerable.  Where  the  saliva  brought  about 
any  diminution  in  the  number  of  micro-organisms,  this  semblance 
of  bactericidal  action  could  be  noted  not  only  in  the  normal  saliva, 
but  also,  as  in  the  lachrymal  secretion  above  described,  in  saliva 

1  "  La  saliva  umana,"  Siena,  1891,  and  Centralbl.f.  Bakteriol  u.  Parasitenk.,  Jena, 
1891,  Bd.  x,  S.  817. 

2  op.  cit. 

3  Ann.  de  Tlnst.  Pasteur,  Paris,  1896,  t.  x,  p.  545. 


416  Chapter  XIII 

heated  to  60°  C.  Against  certain  micro-organisms — the  torulae 
and  the  staphylococci — the  heated  saliva  acted  more  vigorously 
than  did  the  unaltered  saliva.  It  is  consequently  impossible  to 
draw  any  parallel  between  the  action  of  the  saliva  and  that  of  the 
cytases. 

Since  the  saliva  often  contains  (according  to  certain  authors  even 
constantly)  small  quantities  of  potassium  sulphocyanide,  it  seemed  to 
be  worth  while  to  ascertain  whether  this  salt  is  capable  of  destroying 
micro-organisms.  The  experiments  carried  out  by  Hugenschmidt,  in 
order  to  settle  this  point,  demonstrated  that  when  given  in  doses 
comparable  to  those  met  with  in  the  saliva,  the  potassium  sulpho- 
cyanide exerts  no  bactericidal  action. 

Powerless  as  an  antiseptic,  the  saliva  fulfils  an  important  function 
in  ridding  the  mouth  of  micro-organisms  in  a  mechanical  way.  The 
parotid  secretion  and  that  of  the  other  salivary  glands  dilutes  the 
bacteria  and  carries  them  from  the  pharyugeal  cavity  into  the  stomach. 
Hence,  in  diseases  where  the  salivary  secretion  is  much  diminished, 
the  mouth  becomes  the  most  important  portal  of  eiitry  for  micro- 
organisms capable  of  setting  up  secondary  infections.  The  saliva 
is  further  useful  in  diluting  the  alimentary  detritus  and  preventing  its 
stagnation  and  decomposition  in  the  buccal  cavity. 

In  addition  to  the  direct  mechanical  part  played  by  the  saliva, 
it  performs  a  very  important  indirect  function.  This  fluid  con- 
tains microbial  products  and  diastases,  and  is  capable  of  exciting 
in  the  leucocytes  a  positive  chemiotactic  activity.  Hugenschmidt 
demonstrated  the  fact  by  introducing  into  animals  small  capillary 
glass  tubes  containing  saliva.  A  certain  time  after  being  placed  in 
position,  these  tubes  became  filled  with  considerable  masses  of  immi- 
grated leucocytes.  The  same  result  was  obtained  with  guinea-pig's 
[437]  saliva,  enclosed  in  capillary  tubes  and  introduced  into  the  peritoneal 
cavity  of  the  same  species.  Here,  again,  the  leucocytes  assembled  in 
the  tubes  and  ingested  the  micro-organisms  found  in  the  saliva.  The 
influence  of  the  saliva  on  the  afflux  of  the  leucocytes  must  be  regarded 
as  an  act  important  for  the  protection  of  the  buccal  cavity,  and  it  is 
probably  due  to  this  attraction  of  leucocytes  that  lesions  of  this 
region  heal  so  quickly.  The  leucocytes  are  very  numerous  in  the 
glands  of  the  mouth  and  the  tonsils  always  supply  large  quantities 
of  them. 

We  must  not  lose  sight  of  the  fact  that  the  epithelial  covering  of 
the  bucco-pharyngeal  cavity  also  constitutes  an  important  protective 


Immunity  of  the  skin  and  mucous  membranes    417 

factor.  Just  as  on  the  surface  of  the  skin,  the  corneal  cells  are  in  a 
permanent  state  of  desquamation,  so  the  cells  in  the  mouth  are  being 
constantly  renewed.  This  desquamation  increases  especially  during 
mastication,  when  enormous  numbers  of  cells  are  thrown  off;  after 
every  meal  there  is  a  partial  renewal  of  the  surface  of  the  lining 
of  the  buccal  cavity.  Being  covered  on  their  surface,  and  in  their 
interstices  charged  with  innumerable  micro-organisms,  the  epithelial 
cells  carry  away  with  them  all  this  population  from  the  mouth. 

The  numerous  micro-organisms  which  persist  in  the  mouth,  in 
spite  of  all  these  means  for  getting  rid  of  them,  must  also  play 
a  certain  part  in  the  defence  against  infections.  It  is  very  probable 
that  many  of  these  saprophytes  impede  the  multiplication  of  certain 
pathogenic  bacteria ;  but  at  present  it  is  impossible  to  define  more 
exactly  these  phenomena  of  microbial  competition.  It  is  only  because 
we  have  analogies  in  other  regions  of  the  body  that  we  are  able  to 
defend  this  position. 

The  saliva,  incapable  of  destroying  the  micro-organisms  them- 
selves, is  able  to  act  on  their  soluble  products,  as  on  certain  other 
poisons.  In  this  relation  the  action  of  the  saliva  on  snake  venom  is 
most  familiar.  Wehrmann1,  who  has  made  researches  on  this  subject 
in  Calmette's  laboratory  at  Lille,  has  shown  that  the  amylase  (ptyalin) 
of  human  saliva,  mixed  with  very  rapidly  fatal  doses  of  venom,  quite 
prevents  its  toxic  action.  Von  Behring2  reminds  us  on  this  point 
that  the  ancient  Psylli  (a  race  of  northern  Africa),  at  the  beginning  [438] 
of  our  era,  employed  their  saliva  as  an  antidote  against  snake 
bites. 

Powerless  to  kill  the  micro-organisms,  the  saliva  carries  them  off 
mechanically  to  the  exterior  or,  more  frequently,  into  the  stomach. 
The  acid  medium  of  this  great  reservoir  exerts  a  very  marked  effect 
on  these  microscopic  organisms.  It  has  long  been  recognised  that  the 
gastric  juice  prevents  putrefaction  and  can  arrest  it  even  when  it  has 
become  very  advanced.  From  this  observation  an  antiseptic  action  of 
this  juice  was  inferred.  Bacteriological  researches,  undertaken  to 
determine  the  nature  of  this  action,  have  demonstrated  that  several 
species  of  micro-organisms  die  very  shortly  after  being  placed  in  con- 
tact with  the  gastric  juice  in  vitro.  Straus  and  Wurz3  found  that  even 

1  Ann.  de  TInst.  Pasteur,  Paris,  1898,  t.  xn,  p.  510. 

2  "  Allgemeine  Therapie  der  Infectionskrankheiten,"  in  Eulenburg  u.  Samuel's 
"  Lehrb.  d.  allg.  Therapie,"  Berlin  u.  Wien,  1899,  Bd.  in,  S.  980. 

3  Arch,  de  med.  exper.  et  d'anat.  path.,  Paris,  1889, 1. 1,  p.  370. 

B.  27 


418  Chapter  XIII 

anthrax  spores  and  the  tubercle  bacillus  could  be  destroyed  by  gastric 
juice,  after  a  prolonged  sojourn  in  a  sufficient  quantity  of  this  fluid. 
Comparative  researches,  made  with  aqueous  solutions  of  hydrochloric 
acid,  have  demonstrated  that  the  bactericidal  action  of  the  gastric 
juice  depends  solely  on  the  amount  of  this  acid  that  it  contains,  that 
is  to  say,  the  pepsin  plays  no  part  in  the  process.  This  juice  exerts 
no  true  digestive  action  on  the  micro-organisms,  but  it  destroys 
a  certain  number  of  them  by  its  hydrochloric  acid.  This  antiseptic 
action  may  also  be  inferred  from  a  series  of  demonstrations  on  the 
exaggerated  microbial  multiplication  in  cases  where  the  gastric  juice 
has  been  poor  in  hydrochloric  acid.  Several  observers  have  confirmed 
this  bactericidal  action  of  the  gastric  juice  which  is  exerted  specially 
against  certain  species  capable  of  causing  grave  infective  diseases. 
On  the  other  hand,  certain  bacteria  and  other  lower  fungi  are  quite 
resistant  to  the  antiseptic  action  of  this  fluid ;  they  adapt  themselves 
very  readily  to  an  existence  in  the  stomach.  Consequently  there 
exists  in  this  organ,  even  in  animals  such  as  the  dog,  whose  gastric 
juice  contains  most  hydrochloric  acid,  a  special  flora,  whose  most 
characteristic  feature  is  the  relative  insensibility  to  the  acidity 
of  this  medium.  The  Blastoinycetes,  along  with  the  yeasts  and  the 
Torulae,  constitute  the  most  frequent  representatives  of  this  flora; 
alongside  these  may  be  grouped  the  Sarcinae  and  certain  acidophile 
bacilli.  Miller1  has  isolated  several  of  these  micro-organisms  from 
the  contents  of  the  stomach,  and  has  observed  that,  mixed  with  the 
[439]  food,  they  resist  the  action  of  the  gastric  juice,  even  that  of  the  dog, 
whose  hydrochloric  acid  content  is  greater  than  in  man  and  many 
of  the  other  mammals2.  But  these  acidophile  micro-organisms  have 
no  pathogenic  power  and  consequently  are  not  much  to  be  feared. 
It  is  very  doubtful  whether  even  the  infective  bacteria  which  are 
easily  killed  by  the  gastric  juice  m  vitro,  are  often  destroyed  in  the 
stomach.  The  typhoid  coccobacillus,  which  has  shown  itself  to  be  so 

1  Deutsche  med.  Wchnschr.,  Leipzig,  1885,  no.  49. 

2  Amongst  this  acidophile  flora  one  species  merits  particular  attention.     This  is 
a  spirillum,  discovered  by  Bizzozero  in  the  mucous  membrane  of  the  stomach  of  the 
dog.    Salomon  (Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  Jena,  '1896,  Bd.  xix,  S.  433) 
has  studied  this  organism,  not  only  in  the  dog,  but  in  the  cat  and  Norway  rat. 
Multiplying  on  the  mucous  membrane,  the  very  mobile  spirillum  penetrates  into  the 
epithelial  cells  or  is  met  with  inside  vacuoles.    These  latter  being  in  communication 
with  the  external  medium,  the  spirilla  can  readily  penetrate  by  the  openings.     This 
fact  has,  then,  nothing  in  common  with  phagocytosis,  where  it  is  the  cell  which 
ingests  the  micro-organisms  by  means  of  its  amoeboid  movements. 


Immunity  of  the  skin  and  mucous  membranes    419 

sensitive  to  the  destructive  action  of  the  gastric  juice  of  man,  of  the 
dog,  and  of  the  sheep,  is,  from  the  experiments  of  Straus  and  Wurz, 
quite  capable  of  passing  through  the  stomach  without  being  affected. 
Stern1,  as  the  result  of  his  own  researches,  as  well  as  of  those  of  his 
pupils,  came  to  the  conclusion  that  this  micro-organism  is  not  in  the 
least  affected  by  the  gastric  juice  of  a  healthy  man,  containing  the 
normal  amount  of  hydrochloric  acid.  It  was  only  in  cases  of  hyper- 
secretion  and  of  hyperacidity  that  the  micro-organisms  of  typhoid  fever 
were  destroyed  before  they  reached  the  small  intestine. 

The  cholera  vibrio  also  can  pass  through  the  stomach  and  its  acid 
juice.  After  Koch's  demonstration  of  the  great  susceptibility  of  this 
organism  to  acids  in  vitro,  it  was  generally  concluded  that  it  must 
perish  in  the  normal  content  of  the  stomach.  Many  cases  have 
since  been  recorded  in  which  the  cholera  vibrio  was  found,  in  times 
of  cholera  epidemics,  in  the  faeces  of  healthy  persons.  In  order 
to  get  into  the  large  intestine  it  had  to  pass  through  the  normal 
stomach.  In  experimental  cholera  in  young  suckling  rabbits,  a  large 
number  of  vibrios  were  also  found  in  the  distinctly  acid  contents 
of  the  stomach,  and  they  were  seen  to  pass  into  the  small  intestine 
without  any  neutralisation  of  the  acidity  of  the  stomach  taking  place. 
This  example  proves,  once  again,  that  the  phenomena  that  occur 
within  the  living  body  cannot  be  identified  with  those  that  go  on  in 
the  test-tube,  in  vitro. 

Whilst  the  acidity  of  the  gastric  juice  exerts  a  certain  influence  on  [440] 
micro-organisms,  the  pepsin  which  it  contains  acts  unfavourably  on 
their  toxins.  There  are  many  poisons  which  are  readily  absorbed, 
without  being  modified,  by  the  mucous  membrane  of  the  stomach. 
Even  the  venom  of  snakes  can,  under  certain  conditions,  produce 
its  toxic  effect  as  it  is  absorbed  through  the  stomach.  Thus,  ac- 
cording to  the  experiments  of  Wehrmann2,  pepsin  exerts  a  very 
feeble  action  on  this  poison.  On  the  other  hand,  this  diastase  has 
a  marked  action  on  certain  bacterial  toxins.  Gamaleia3  pointed  out 
that  pepsin  destroys  the  diphtheria  toxin.  Charrin  and  Lefevre* 
have  shown  that  it  also  weakens  other  microbial  toxins.  According 
to  Nencki  and  Mmes  Sieber  and  Schoumow-Simanowski5,  the  gastric 

1  Von  Volkmann's  Samml.  klin.  Vortr.,  Leipzig,  1898,  no.  38,  S.  290. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1898,  t.  xn,  p.  510. 

3  Compt.  rend.  Soc.  de  biol.,  Paris,  1892,  p.  153. 

4  Compt.  rend.  Soc.  de  biol.,  Paris,  1897,  p.  830,  and  Charrin,  "Les  defenses 
naturelles  de  I'organisme,"  Paris,  1898,  p.  128. 

5  CentralU.f.  Bakteriol.  u.  Parasitenk.,  Jena,  1898,  Bd.  xxni,  SS.  840,  880. 

27—2 


420  Chapter  XIII 

juice  of  the  dog  destroys  relatively  small  quantities  of  the  diphtheria 
poison.  A  gramme  of  the  juice  is  capable  of  rendering  innocuous 
50  lethal  doses  of  this  toxin,  but,  in  order  that  this  action  may  be 
produced,  a  prolonged  contact  of  the  two  substances  is  required. 
Since  the  neutralised  gastric  juice  acts  in  the  same  way,  this  effect 
must  be  attributed  not  to  the  acidity  of  the  gastric  juice,  but  rather 
to  the  amount  of  pepsin  it  contains.  This  diastase  acts  much  more 
powerfully  on  the  tetanus  toxin,  1  gramme  of  gastric  juice  neutral- 
ising 10,000  doses  lethal  for  the  guinea-pig.  On  the  other  hand, 
abrin  is  not  modified  by  the  gastric  juice  according  to  the  researches 
of  Repin1,  carried  out  in  Roux's  laboratory.  Nevertheless,  its  action 
when  administered  by  the  stomach  is  feeble,  and  Ehrlich2  has  been 
enabled  to  vaccinate  small  animals  against  this  vegetable  poison  by 
availing  himself  of  his  knowledge  of  this  fact.  Repin  explains  this 
result  as  due  to  the  very  slight  absorption  of  abrin  by  the  gastro- 
intestinal mucous  membrane.  This  same  factor,  Re"pin  thinks,  may 
contribute  also  to  the  failure  of  various  toxins  when  ingested.  This 
rule,  however,  is  not  an  absolute  one.  Thus,  the  toxin  of  the  botu- 
linic  bacillus  of  van  Ermengem3  is  not  destroyed  by  the  digestive 
diastases,  and  it  is  certainly  absorbed  by  the  mucous  membrane  of 
the  alimentary  canal.  For  this  reason,  when  it  is  introduced  by  way 
of  the  stomach,  it  exhibits  a  very  violent  toxic  activity. 

The  stomach,  though  capable,  through  its  acid,  of  preventing 

[441]  the  multiplication  of  certain  micro-organisms,  protects,  very  feebly, 

the  rest  of  the  digestive  apparatus.    As  soon  as,  in  the  duodenum, 

the  acidity  is  weakened  or  neutralised,  the  various  micro-organisms 

commence  to  multiply  and  soon  develop  very  abundantly. 

In  the  animal  series  the  intestine  proper  presents  a  very  great 
variability,  and  even,  in  closely  allied  species,  exhibits  considerable 
differences.  From  the  particular  point  of  view  which  interests  us 
these  differences  are  very  marked.  Alongside  insects,  such  as  the 
silkworm,  the  larvae  of  cockchafers  and  others,  whose  intestinal  canal 
contains  a  very  rich  bacterial  vegetation,  we  have  others  which  contain 
exceedingly  few  micro-organisms  or,  indeed,  none  at  all.  This  last 
condition  is  represented  by  the  caterpillars  of  small 'Lepidoptera,  and 
notably  by  those  of  several  species  of  clothes-moths.  These  differences 
correspond  to  the  variety  of  the  juices  and  digestive  ferments  met  with 

1  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  517. 

2  Deutsctie  med.  Wchnschr.,  Leipzig,  1891,  SS.  976,  1218. 

3  Centralbl.f.  Bakteriol  u.  Parasitenk.,  Jena.  1896,  Bd.  xix,  S.  442. 


Immunity  of  the  skin  and  mucous  membranes    421 

in  these  Invertebrata.  As  the  physiology  of  digestion  in  these  animals 
is  as  yet  little  understood,  it  is  at  present  impossible  to  define  clearly 
the  conditions  which  regulate  these  phenomena.  In  any  case,  it  is 
very  probable  that  the  soluble  digestive  ferments  destroy  the  micro- 
organisms and  prevent  them  from  growing  in  the  intestinal  content. 
Otherwise  it  is  difficult  to  explain  why  the  larvae  of  clothes-moths, 
which  live  in  old  dusty  textile  fabrics,  where  the  germs  of  bacteria  are 
not  wanting,  present  a  digestive  canal  from  which  micro-organisms 
are  entirely  absent.  The  digestive  juices,  adapted  to  digest  wool 
and  even  wax,  are  evidently  capable  also  of  digesting  the  bodies  of 
micro-organisms.  In  other  insects,  which  feed  on  vegetables  and  on 
substances  less  difficult  to  digest,  micro-organisms  develop  in  the 
intestinal  content,  as  in  many  of  the  higher  animals.  Insects  often 
have  their  intestine  lined  by  a  very  delicate  chitinous  membrane 
which  offers  no  obstacle  to  the  absorption  of  the  products  of  digestion, 
but  prevents  the  micro-organisms  from  reaching  the  epithelial  layer. 
We  have  here  a  defensive  apparatus  against  microbial  invasion,  which 
must  be  the  more  useful  because  this  membrane  is  thrown  off  and 
renewed  at  each  moult,  thus  enabling  the  insect  to  rid  itself  at  one 
swoop  of  a  large  number  of  its  microscopic  inhabitants. 

In  the  Vertebrata  the  canal  of  the  pancreas  and  that  of  the  small 
intestine  are  always  populated  by  a  greater  or  smaller  number  of 
micro-organisms,  amongst  which  bacilli  predominate.  We  know  the 
great  difficulty  experienced  every  time  we  wish  to  make  experiments 
on  the  pancreatic  digestion  outside  the  animal  body.  The  digestive  [442] 
fluid,  alkaline  and  containing  many  bacteria,  is  soon  transformed 
into  a  microbial  purge.  We  are  then  obliged  to  have  recourse  to 
antiseptics  to  arrest  this  development  and  to  bring  into  prominence 
the  digestive  role  played  by  the  soluble  ferments  of  the  pancreas. 
This  well-known  fact  may  be  used  as  an  argument  against  the 
existence  of  any  kind  of  bactericidal  power  in  the  small  intestine 
of  higher  vertebrates.  Even  in  those  animals  which  are  distin- 
guished by  the  remarkable  poorness  of  their  intestinal  flora,  we  fail 
to  reveal  the  presence  of  bactericidal  substances.  The  Crustacea, 
e.g.  the  crayfish,  and  certain  worms,  such  as  the  Ascaris,  contain  few 
micro-organisms  in  their  intestine.  The  former  feed  on  putrescent 
substances,  the  latter  inhabit  the  small  intestine  of  man  and  animals, 
populated  by  myriads  of  bacteria.  It  might  be  supposed  that,  under 
these  conditions,  the  intestinal  content  must  contain  a  mass  of  micro- 
organisms or,  if  that  be  not  the  case,  that  it  must  contain  some 


422  Chapter  XIII 

substance  which  is  powerfully  bactericidal.  In  reality,  neither  one 
nor  the  other  of  these  suppositions  receives  any  confirmation.  The 
intestines  of  the  two  Invertebrata  I  have  named  are  very  poor  in 
micro-organisms  and  their  contents  do  not  exhibit  the  slightest 
bactericidal  power.  When  a  little  of  their  contents  is  placed  in 
tubes  and  kept  at  a  suitable  temperature  it  is  not  long  before  it 
becomes  filled  by  a  great  number  of  bacteria  of  various  kinds. 

To  explain  this  poverty  of  the  microbian  flora  of  the  intestines  in 
these  examples  we  must  postulate  some  kind  of  mechanical  purifi- 
cation, facilitated  by  the  peristaltic  movements  of  the  digestive  canal. 
Even  in  animals  which  have  an  abundance  of  micro-organisms 
in  the  small  intestine,  there  must  be  produced  some  phenomenon 
which  brings  about  the  disappearance  of  a  certain  number  of  them. 
In  mammals  the  small  intestine  always  contains  far  fewer  micro- 
organisms than  does  the  large  intestine  ;  in  birds,  the  coecum  is  much 
richer  in  bacteria  than  is  the  rest  of  the  digestive  canal.  Schiitz1  has 
attempted  to  demonstrate  the  disinfecting  power  of  the  small  intestine 
in  the  dog  by  feeding  it  on  substances  to  which  he  had  added  a  large 
number  of  Gamaleia's  vibrio  ( Vibrio  metchnikovi).  After  convincing 
himself  that  micro-organisms  perish  in  the  digestive  canal  and  are 
never  found  in  the  excrementa,  Schiitz  introduced  into  his  dogs 
[443]  a  cannula,  one  branch  of  which  passed  into  the  pylorus,  the  other 
into  the  duodenum.  By  means  of  a  small  apparatus  he  could  readily 
interrupt  the  communication  between  the  stomach  and  the  intestine. 
The  vibrios,  mixed  with  biscuit,  and  softened  with  water,  introduced 
directly  into  the  duodenum  (whilst  the  stomach  was  kept  completely 
isolated),  penetrated  into  the  large  intestine  in  small  numbers  only. 
The  lower  part  of  the  colon,  the  rectum  and  the  excrements  gave  no 
cultures  of  vibrios  and  did  not  give  rise  to  any  growth  except  that  of 
the  Bacillus  coli.  In  this  case  the  disinfection  of  the  intestine  took 
place  without  any  help  from  the  gastric  juice.  Further,  when  Schiitz 
killed  dogs,  after  giving  them  food  in  which  vibrios  were  mixed,  these 
organisms  were  found  in  the  intestine  only.  The  gastric  acidity, 
therefore,  is  not  capable  of  killing  these  organisms,  or  of  preventing 
them  from  passing  into  the  small  intestine,  in  which, alone  they  were 
killed.  It  was  only  with  the  aid  of  purgatives,  such  as  castor-oil  or 
calomel,  that  Schiitz  succeeded  in  preserving  the  vibrios  in  the  intes- 
tines and  in  finding  them  in  the  dejecta.  This  observer  did  not  carry  his 
investigations  further  and  did  not  make  out  the  mechanism  by  which 
1  Berl.  klin.  Wchnschr.,  1900,  S.  553. 


Immunity  of  the  skin  and  mucous  membranes    423 

the  small  intestine  destroyed  such  large  numbers  of  vibrios.  He 
supposes  that  alongside  a  mechanical  factor,  such  as  the  very  active 
peristaltic  movement,  there  exist  others,  perhaps  chemical  processes, 
capable  of  killing  these  micro-organisms. 

This  question  of  the  defensive  action  in  the  small  intestine  is, 
consequently,  far  from  being  settled.  The  data  collected  indicate 
merely  that  the  problem  is  a  very  complex  one.  It  has  been  shown, 
however,  that  very  virulent  bacteria  may  pass  through  the  digestive 
canal  not  only  without  injuring  the  animal  but  even  meeting  their 
own  death  in  this  organ.  The  anthrax  bacillus,  so  fatal  to  mice  and 
guinea-pigs,  may  be  swallowed  by  these  animals  without  the  slightest 
danger  to  them.  It  may  then  be  found  in  the  small  intestine,  but  not 
in  the  large  intestine,  this  proving  that  the  gastric  acidity  is  incapable 
of  destroying  them  outright  To  produce  generalised  anthrax  by 
way  of  the  intestine,  it  was  necessary  that  the  animals  should  swallow 
the  spores  of  anthrax  along  with  spiny  plants,  as  in  the  experiments 
of  Pasteur  and  his  collaborators1,  or  along  with  sand  or  powdered 
glass.  In  these  cases  the  intestinal  lesions  served  as  the  port  of 
entry  for  the  bacillus,  the  intact  mucous  membrane  of  the  intestine  [444] 
preventing  their  penetration.  Mitchell,  in  an  unpublished  work, 
undertaken  in  my  laboratory,  succeeded  in  giving  fatal  anthrax  to 
guinea-pigs,  even  when  he  fed  them  with  spores  mixed  with  the 
"  crumb  "  of  bread  soaked  in  milk.  During  the  whole  period  of  the 
experiment  the  animals  took  no  food  capable  of  producing  lesions 
of  the  wall  of  the  intestine.  But  examples  of  infection  under  these 
conditions  are  altogether  exceptional.  In  the  great  majority  of 
instances  the  animals  were  not  attacked.  The  same  rule  applies  also 
to  many  other  micro-organisms,  which  can  be  ingested  with  impunity 
although  their  inoculation  into  the  blood  and  tissues  sets  up  infections 
which  are  inevitably  fatal.  Many  animals  may,  without  running  the 
least  ri:*k,  swallow  large  numbers  of  bacteria  which  in  man  produce 
grave  intestinal  disease.  Thus,  it  has  never  been  possible  to  produce 
typhoid  fever  regularly  and  with  certainty  in  any  of  the  species  of 
animals  to  which  masses  of  typhoid  coccobacilli  were  given  by  in- 
gestion.  We  may  recall  the  difficulties  which  so  many  investigators 
have  met  with  in  inducing  intestinal  cholera  in  laboratory  animals, 
which  are  so  refractory  to  Koch's  vibrio.  Only  very  young  animals, 
especially  unweaned  rabbits,  are  capable  of  contracting  fatal  intestinal 
cholera,  but  these  animals  may  contract  it  not  only  from  the  true 
1  Compt.  rend.  Acad.  d.  sc.,  Paris,  1880,  t.  xci,  p.  86. 


424  Chapter  XIII 

cholera  vibrio  but  also  from  Gamaleia's  vibrio.  As  soon  as  rabbits 
begin  to  feed  on  vegetables  they  acquire  an  immunity  which  is 
insuperable. 

It  is  most  assuredly  not  the  digestive  ferments  of  the  intestine 
that  protect  the  animal  against  infection  through  the  intestine. 
The  contents  of  every  part  of  the  small  intestine  of  the  Vertebrata 
permit  an  abundant  development  of  all  sorts  of  bacteria,  and  in 
solutions  of  trypsin  not  only  do  pathogenic  and  resistant  micro- 
organisms grow  luxuriantly,  but  also  saprophytes  and  the  most 
inoffensive  bacteria.  Weigert1  influenced  by  this  fact  even  saw  in  it 
an  objection  to  the  theory  that  the  destruction  of  micro-organisms 
in  the  animal,  notably  that  which  is  effected  by  the  phagocytes,  is  to 
be  regarded  as  an  act  of  digestion.  It  is  a  remarkable  fact  that 
whilst  trypsin  is  so  powerless  against  micro-organisms  the  intra- 
cellular  ferments,  and  especially  micro-cytase,  whose  kinship  with  the 
group  of  trypsins  is  undeniable,  are  able  to  bring  about  their  digestion 
so  completely. 

[445]  It  was  thought  that  among  the  digestive  fluids  the  bile  more 
especially  exerts  a  definite  antiseptic  power.  It  is  undeniable  that 
this  fluid  is  not  indifferent  for  certain  bacteria.  Talma  affirms 
that  it  is  bactericidal  for  several  micro-organisms,  especially  the 
diphtheria  bacillus.  In  many  of  his  experiments,  however,  the  bile 
proved  to  be  incapable  of  killing  micro-organisms  introduced  directly 
into  the  gall-bladder.  According  to  the  researches  of  Gilbert  and 
Dominici2  the  bile  does  not  prevent  the  abundant  development  of 
micro-organisms  capable  of  setting  up  diseases  of  the  biliary  passages, 
such  as  the  Bacillus  coli.  I  have  tried  to  prevent  the  multiplication 
of  the  cholera  vibrio  by  the  addition  of  bile,  but  my  results  were 
entirely  negative.  If  the  bile  in  an  undiluted  state  has  such  a  slight 
action  upon  so  many  kinds  of  bacteria,  it  is  evident  that  we  cannot 
count  upon  its  antiseptic  action  when  it  passes  into  the  small 
intestine,  where  it  is  mixed  with  all  sorts  of  other  substances. 

The  digestive  fluids  of  the  small  intestine,  either  those  that  are 
non-bactericidal,  the  pancreatic  juice,  or  those  that  are  not  very 
active,  the  bile,  are,  nevertheless,  capable  of  producing  a  marked 
influence  on  certain  poisons,  and  amongst  others  on  certain  microbial 
toxins.  According  to  the  experiments  of  Nencki  and  of  Mmes  Sieber 
and  Schoumow-Simanowski  (I.e.),  trypsin  is  much  more  antitoxic 

1  Fortschr.  d.  Med.,  Berlin,  1888,  Bd.  vi,  S.  809. 
*  Compt.  rend.  Soc.  de  biol.,  Paris,  1894,  p.  38. 


Immunity  of  the  skin  and  mucous  membranes    425 

against  the  diphtheria  poison  than  is  pepsin.  Thus,  the  pancreatic 
juice  of  both  the  rabbit  and  the  guinea-pig  destroys  this  toxin  much 
more  actively  than  does  the  gastric  juice.  The  pancreatic  juice  of  the 
dog  exerts  a  very  powerful  action  on  the  same  toxin.  A  gramme  of  this 
fluid  neutralises  ten  thousand  lethal  doses  of  the  toxin.  Wehrmann, 
also,  found  that  trypsin  inhibits  the  poisonous  action  of  snake 
venom.  Bile  also  exerts  an  action  upon  certain  poisons.  Mixed 
with  diphtheria  and  tetanus  toxins  it  prevents  their  pathogenic  effect. 
It  also  neutralises  the  venom  of  snakes,  as  has  been  observed  by 
Fraser1,  Phisalix2  and  Calmette3.  All  the  venoms,  when  placed  in 
contact  with  fresh  bile  for  24  hours,  induce  no  injurious  effect  when 
the  mixture  is  injected  into  normal  animals.  Bile,  heated  to  100°  C.,  [446] 
and  even  to  120°C.,  is  still,  though  more  feebly,  active.  To  obtain 
these  results,  however,  it  is  indispensable  to  prepare,  beforehand, 
a  mixture  of  the  two  fluids.  When  injected  separately,  whether  at 
the  same  time  as,  before,  or  after,  the  venom,  the  bile  does  not 
prevent  poisoning.  The  venom  when  injected  directly  into  the  gall- 
bladder of  the  rabbit  sets  up  fatal  intoxication  to  the  same  degree  as 
does  the  same  dose  of  venom  introduced  subcutaneously.  Calmette, 
who  made  this  experiment,  explains  this  negative  result  as  due  to  the 
too  rapid  absorption  of  the  venom,  which  has  not  had  time  to  be 
affected  by  the  destructive  action  of  the  bile. 

A  protective  action  of  the  bile  has  been  determined  with  regard 
to  two  viruses,  the  micro-organisms  producing  which  are  not,  as  yet, 
known.  Koch4  succeeded  in  vaccinating  Bovidae  with  the  bile  of 
animals  that  had  died  from  rinderpest,  and  Frantzius5  prevented 
animals  from  contracting  rabies  when  he  inoculated  into  them  rabic 
virus  mixed  with  the  bile  of  rabbits  that  had  succumbed  to  that 
disease.  Vallee6  points  out,  however,  that  the  bile  of  the  normal 
rabbit  produces  exactly  the  same  effect.  Here,  then,  we  have  to  do 
with  a  preventive  action  of  the  bile,  as  such,  against  the  rabic  virus. 
In  the  present  state  of  our  knowledge  it  is  impossible  to  say  whether 
this  influence  of  the  bile  is  directed  against  the  toxin  or  against  the 
unknown  micro-organism.  Analogy  would  lead  us  to  accept  the 
former  of  these  two  suppositions. 

1  Brit.  Med.  Journ.,  London,  1897,  VoL  II,  p.  595. 

2  Compt.  rend.  Soc.  de  biol,  Paris,  1898,  p.  1057. 

3  Ann.  de  VInst.  Pasteur,  Paris,  1898,  t.  xn,  p.  345. 

4  Deutsche  med.  Wchnschr.,  Leipzig,  1897,  SS.  225,  241. 

5  Centralbl  f.  Bakteriol  u.  Parasitenk.,  Jena,  1898,  Abt.  I,  Bd.  xxm,  S.  782. 

6  Ann.  de  Vlnst.  Pasteur,  Paris,  1899,  t  xiu,  p.  506. 


426  Chapter  XIII 

The  bile,  active  against  certain  poisons,  does  not,  however,  prevent 
poisoning  by  cholera  toxin  nor  by  that  of  botulism,  two  most  typical 
intestinal  intoxications. 

Since  diastases  and  the  digestive  juices  are  incapable  of  affecting 
micro-organisms  and  since  certain  of  these  latter  perish  in  the  intes- 
tines we  must  seek  some  other  cause  for  their  destruction.  It  is 
probable  that  the  vital  competition  among  the  micro-organisms, 
whose  r61e  could  be  foreseen  in  the  buccal  cavity,  is  of  still  greater 
importance  in  relation  to  the  phenomena  of  pathogenic  action  or  of 
the  innocuousness  of  infective  bacteria  in  the  intestinal  canal1.  This 
[447]  complex  and  difficult  chapter,  up  to  the  present,  has  been  studied 
in  a  very  imperfect  fashion.  In  our  observations  on  cholera  we 
have  remarked  that  under  certain  conditions  the  cholera  vibrios  do 
not  develop  on  gelatine  plates,  except  in  the  neighbourhood  of 
certain  adjuvant  micro-organisms  such  as  the  Torulae  and  the 
Sarcinae.  Guided  by  this  fact  we  have  succeeded  in  producing 
intestinal  cholera  in  suckling  rabbits,  with  races  of  vibrios  which, 
when  ingested  alone  by  these  animals,  remain  innocuous  or  set  up 
the  disease  only  occasionally.  We  have  convinced  ourselves  of  the 
helpful  action  of  certain  representatives  of  the  gastro-intestinal  flora 
upon  true  cholera2.  Following  on  these  observations,  it  was  quite 
natural  to  suppose  that  this  flora  might  also  contain  micro-organisms 
capable  of  hindering  the  development  and  toxic  action  of  the  cholera 
vibrio.  We  have  even  advanced  the  hypothesis  that  these  "hindering" 
micro-organisms  in  the  flora  of  the  digestive  canal  may  explain  the 
immunity  of  animals,  of  many  human  individuals,  and  even  of  the 
population  of  unattacked  towns,  to  intestinal  cholera.  We  should 
have,  then,  in  the  intestinal  contents,  inhabited  by  a  number  of 
micro-organisms  and  deprived  of  bactericidal  juices,  an  important 
factor  which  in  many  cases  guarantees  a  refractory  condition.  It 
must  be  stated,  however,  that  prolonged  studies,  carried  out  with 

1  Perhaps  the  intestinal  micro-organisms  also  play  a  part  in  the  immunity  of  the 
animal  against  Entozoa.    Many  of  the  examples  of  this  immunity  are  very  striking. 
Certain  intestinal  worms  can  live  only  in  the  digestive  canal  of  a  single  or  of  a  very 
small  number  of  species  of  animals.    When  we  feed  rabbits  with  a  quantity  of  the 
cysticerci  of  the  pig  these  pass  living  into  the  small  intestine  and  are  there  trans- 
formed into  true  scolices.    But,  instead  of  reproducing  themselves,  they  are  expelled 
and  never  give  rise  to  the  development  of  taeniae.    The  immunity  against  intestinal 
parasites  has  never  been  made  the  object  of  special  study,  and  it  is  only  as  a  pure 
hypothesis  that  I  offer  this  suggestion  as  to  the  part  played  by  the  micro-organisms 
of  the  intestinal  flora. 

2  Ann.  de  VInst.  Pasteur,  Paris,  1894,  t.  vm,  p.  549. 


Immunity  of  the  skin  and  mucous  membranes    427 

the  object  of  demonstrating  in  suckling  rabbits  the  precise  part 
played  by  these  micro-organisms  which  prevent  cholera,  have  not 
given  any  satisfactory  results.  This  we  attribute  to  our  very  im- 
perfect knowledge  of  the  microbial  population  of  the  digestive  organs. 

If  the  destruction  by  representatives  of  the  normal  intestinal  flora 
of  the  micro-organisms  which  penetrate  into  the  intestines  has  not  as 
yet  been  satisfactorily  demonstrated,  the  power  of  these  latter  to 
destroy  microbial  toxins  cannot  be  doubted.  We1  have  shown  that 
a  great  number  of  micro-organisms  develop  well  in  broth  cultures  of 
the  tetanus  bacillus  which  contain  a  quantity  of  specific  toxin.  This 
toxin  is  destroyed  under  the  influence  of  this  microbial  vegetation, 
but  the  production  of  antitoxin  never  results.  Charrin  and  Mangin2  [448] 
have  observed  similar  facts. 

As  the  destruction  of  bacterial  toxins  by  micro-organisms  takes 
place  with  great  constancy  and  rapidity,  it  is  quite  natural  to  suppose 
that  the  same  phenomenon  occurs  also  in  the  intestinal  canal  of  living 
animals  in  which  pathogenic  micro-organisms  have  succeeded  in 
secreting  their  toxic  products. 

The  liver  having  long  been  recognised  as  the  purifying  organ 
of  the  products  resulting  from  digestion,  it  has  been  asked  if  it  might 
not  also  play  a  part  in  the  destruction  of  microbial  poisons.  Certain 
facts  point  to  its  inhibiting  influence  on  the  action  of  nicotin,  atropin, 
and  of  certain  other  alkaloids,  and  we  have  other  facts  which 
demonstrate  the  power  of  the  liver  to  transform  into  urea  the 
ammoniacal  substances  arising  from  the  activity  of  the  digestive 
glands.  When  Nencki,  Pawloff,  and  their  collaborators3  succeeded  in 
making  the  portal  vein  communicate  with  the  vena  cava,  and  thus 
were  able  to  suppress  the  purifying  function  of  the  liver,  they  found 
that  their  dogs  became  poisoned  in  consequence  of  the  accumulation 
of  ammonia  in  the  animal  organism. 

Guided  by  these  data  as  to  the  protective  r6Ie  played  by  the 
liver  an  attempt  was  made  to  apply  them  to  the  action  of  this  organ 
on  bacterial  toxins  such  as  the  diphtheria  poison.  The  numerous 
attempts  undertaken  in  this  direction  have  given  negative  results : 
the  liver  was  not  found  to  be  capable  of  destroying  this  toxin. 
Bouchard,  Charrin  and  Buffer  have  studied  the  action  of  the  liver  on 
the  pyocyanic  toxin.  They  thought  that  they  could  make  out  a 

1  Ann.  de  I'lnst.  Pasteur,  Paris,  1897,  i.  xi,  p.  802. 

2  Compt.  rend.  Soc.  de  biol.,  Paris,  1897,  p.  545. 

3  Arch,  de  Sci.  biol.,  St  Petersbourg,  1892,  1. 1. 


428  Chapter  XIII 

certain  antitoxic  action  of  this  organ,  but,  later,  Charrin1  convinced 
himself  that  the  bacterial  secretions  are  only  "  moderately  modified  " 
under  these  conditions,  and  that  it  is  more  especially  the  parts  soluble 
in  alcohol  which  undergo  modification  in  the  liver.  Now,  the  true 
bacterial  toxins,  as  is  well  known,  are  distinguished  by  their 
insolubility  in  alcohol.  Moreover  in  the  numerous  experiments  made 
by  Roux  and  Vaillard  and  so  many  other  observers  on  the  tetanus 
and  diphtheria  toxins  there  has  never  been  any  evidence  of  any  kind 
of  antitoxic  action  of  the  liver. 

The  digestive  organs  are  furnished  throughout  with  a  defensive 
[449]  apparatus  against  micro-organisms  ;  this  consists  in  an  accumulation 
of  lymphoid  tissue  in  the  form  of  patches  or  groups  of  solitary 
glands : — the  tonsils,  Peyer's  patches,  and  the  solitary  glands  of 
the  intestine.  These  organs  produce  a  large  number  of  phagocytes 
which  are  able  to  come  into  close  contact  with  the  micro-organisms. 
Ribbert2  and  Bizzozero3  have,  independently  or  almost  simultaneously, 
described  glandular  masses  in  the  coecum  of  the  rabbit  in  which  they 
recognise  the  presence  of  many  micro-organisms  derived  from  the 
intestinal  content.  They  noted  that  the  greater  number  of  these 
bacteria  were  within  cells,  and  regarded  this  as  an  example  of 
phagocytic  reaction.  Manfredi4  was  able  to  confirm  this  interpretation 
by  the  demonstration  that  the  ingested  micro-organisms  were  dead. 
Later,  Buffer5  studied  this  question  in  my  laboratory.  He  observed 
intestinal  phagocytosis  in  Peyer's  patches  in  several  species  of  animals, 
and  showed  that  the  lymphoid  tissue  contained  large  macrophages 
filled  with  bacteria  and  microphages  in  process  of  intracellular 
digestion.  Amongst  these  latter  he  recognised  leucocytes,  which  in 
turn  contained  micro-organisms.  The  accumulation  of  phagocytes 
in  the  lymphoid  organs  of  the  digestive  canal  constitutes,  so  to  speak, 
the  last  act  of  a  struggle  which  is  spread  over  a  very  wide  field. 

Some  years  ago  Stohr  demonstrated6  that  the  wall  of  the  intestine, 
and  especially  the  tonsils  and  other  lymphoid  organs,  are  traversed 
by  an  enormous  number  of  leucocytes  which  execute  a  kind  of 
migration  towards  the  cavities  containing  micro-organisms.  This  con- 

"  Les  defenses  naturelles  de  1'organisme,"  Paris,  1898. 

2  Deutsche  med.  Wchnschr.,  Leipzig,  1885,  S.  197. 

3  Centralb.f.  d.  med.  Wissensch.,  Berlin,  Jahrg.  1885,  S.  801. 
*  Gior.  internaz.  d.  sc.  med.,  Napoli,  1886,  p.  318. 

6  Quart.  Journ.  Micr.  Sc.,  Lond.,  1890,  Vol.  xxx,  n.s.,  p.  481. 
0  Virchow's  Archiv,  1884,  Bd.  xcvn,  S.  211. 


Immunity  of  the  skin  and  mucous  membranes    429 

tinual  and  normal  condition  is  often  termed  Stohr's  phenomenon. 
It  is  evident  that  we  have  here  a  process  of  phagocytic  defence  in 
which  the  leucocytes,  disseminated  through  the  digestive  canal,  give 
chase  to  the  micro-organisms  that  are  nearest  to  the  living  portions 
of  this  organ.  When  we  remove  a  particle  of  mucus  from  the 
surface  of  the  tonsils  of  a  person  in  good  health  we  always  find 
that  it  contains  leucocytes,  especially  microphages,  filled  with 
micro-organisms  of  all  kinds. 

The  protection  of  the  digestive  mucous  membrane  is  a  more 
complicated  process  than  that  of  other  mucous  membranes,  and  many  [450] 
of  the  points  concerned  therein  are  still  obscure  and  need  to  be 
elucidated  by  further  research.  It  might  be  thought  that  the  pheno- 
mena, associated  with  the  defence  of  the  mucous  membrane  of  the 
genital  organs,  being  much  more  simple  and  yet  of  similar  nature, 
should  be  much  more  easily  made  out,  and  that  these  would  throw 
light  on  several  aspects  of  the  problem  of  the  general  defence  of 
the  animal.  Obstetricians  and  gynaecologists  have  certainly  given 
much  attention  to  this  question  as  regards  the  female  genital  organs, 
but  we  are  still  far  from  possessing  a  satisfactory  knowledge  of  this 
subject.  There  already  exists  quite  a  literature  on  the  question, 
dominated  by  the  work  in  two  volumes  published  by  Menge  and 
Kronig1,  but  a  satisfactory  solution  has  still  to  be  obtained. 

At  birth  the  vulva  and  the  vagina  are  free  from  micro-organisms, 
but  they  soon  become  inhabited  and  a  fairly  abundant  flora,  in 
which  may  be  recognised  certain  predominant  species,  such  as  the 
bacillus  of  Doederlein,  is  developed.  Micro-organisms,  therefore, 
can  exist  in  the  vulva  and  the  vagina,  and  yet,  when  we  intro- 
duce into  these  organs  cultures  of  various  bacteria,  saprophytic 
or  pathogenic,  they  soon  disappear.  We  have  the  phenomenon  to 
which  Menge  has  given  the  name  of  "  autopurification  "  of  the  female 
genital  organs.  He  himself,  as  well  as  his  predecessors,  Doederlein 
and  Stroganoff,  tried  to  make  out  the  mechanism  of  this  purification. 
In  the  new-born  female  child  the  phenomenon  is  less  complicated 
than  in  the  adult.  According  to  Menge  the  acidity  of  the  vaginal 
secretion  in  these  infants  at  first  prevents  the  development  of  a 
large  number  of  bacteria.  Associated  with  this  factor  is  a  marked 
emigration  of  leucocytes,  which  destroy  the  bacteria  by  an  act  of 
phagocytosis,  or  perhaps  by  their  products  that  have  escaped  into 
the  vaginal  mucus.  As  a  third  element  to  which  much  importance 
1  "  Bakteriologie  des  weiblichen  Genitalkanals,"  Leipzig,  1897. 


430  Chapter  XIII 

is  attributed,  we  must  accept  the  intervention  of  acidophile  bacteria 
which  grow  well  in  acid  secretions  but  which  hinder  the  development 
of  other  micro-organisms.  Doederlein  concludes  that  it  is  more 
especially  to  the  bacillus  which  bears  his  name  that  the  vagina  owes 
its  protection  against  infective  germs.  Menge,  however,  attributes 
this  action  to  a  whole  series  of  bacteria. 

After  introducing  a  quantity  of  the  StapJiylococcus  pyogenes 
into  the  vagina  of  new-born  females,  Menge  found  that  they  grew 
[451]  for  a  certain  length  of  time.  Their  presence  excited  a  great  ac- 
cumulation of  leucocytes  in  the  vaginal  mucus,  this  being  followed 
by  a  very  marked  ingestion  of  the  micro-organisms,  but  it  was  only 
from  the  moment  when  the  vagina  became  peopled  with  the  bacteria 
which  constitute  its  normal  flora  that  the  staphylococci  began  to 
disappear.  This  process  of  autopurification  only  ceased  three  days 
after  the  introduction  of  these  bacteria.  Menge  asked  himself 
whether  some  purely  mechanical  element  did  not  contribute  to  rid 
the  vagina  of  the  micro-organisms  which  had  entered  it.  To  settle 
this  point  he  introduced  into  this  cavity  grains  of  vermilion,  and 
as  these  latter  remained  there  for  a  longer  period  than  did  the 
micro-organisms,  he  concluded  that  the  vagina  was  incapable  of 
purifying  itself  by  mechanical  means.  We  must,  however,  in  these 
experiments  take  into  account  the  fact  that  the  micro-organisms 
which  Menge  introduced  into  the  vagina  excited  considerable  reaction, 
accompanied  by  a  marked  leucocytosis.  Under  these  conditions 
there  should  be  produced  a  greater  quantity  of  the  mucous  secretions 
which  could  much  more  readily  carry  off  with  them  the  micro- 
organisms that  had  come  into  the  vagina  than  the  smaller  quantity 
could  deal  with  the  vermilion.  It  is  very  probable,  therefore,  that, 
just  as  in  the  case  of  the  other  mucous  membranes,  that  of  the 
female  genital  organs  is  capable  of  mechanically  expelling  fine 
particles,  and  especially  micro-organisms. 

With  the  object  of  throwing  further  light  on  the  problem  of  the 
autopurification  of  the  vagina,  Cahanescu1,  working  in  my  laboratory, 
undertook  experiments  on  the  females  of  several  species  of  mammals. 
The  mare,  as  producing  the  greatest  amount  of  vaginal  mucus,  was 
selected  by  this  observer  as  suitable  for  the  settling  of  this  question 
of  the  bactericidal  power  of  this  secretion.  The  result  was  absolutely 
negative,  even  when  such  an  inoffensive  saprophyte  as  the  Cocco- 
bacillus  prodigiosus  was  used.  The  autopurification  of  the  vagina 
1  Ann.  de  I'Inst.  Pasteur,  Paris,  1901,  t.  xv,  p.  842. 


Immunity  of  the  skin  and  mucous  membranes    431 

of  the  female  dog,  rabbit  and  guinea-pig,  was  found  to  be  neither 
very  marked  nor  very  active.  The  micro-organisms  introduced  into 
the  vagina  usually  remained  there  for  some  time.  Of  all  the  factors 
in  the  microbial  destruction  which  Cahanescu  was  able  to  make  out 
that  of  the  accumulation  of  leucocytes  was  the  most  active.  Some- 
times he  observed  an  extraordinary  amount  of  phagocytosis,  whilst 
in  other  experiments  this  was  slight  or  even  absent.  Many  of  the 
leucocytes  being  killed  in  the  vaginal  mucus,  it  is  possible  that  in 
some  cases  a  certain  bactericidal  action  of  the  cytases  which  have  [452] 
escaped  from  these  dead  leucocytes  is  set  up.  It  is  true  that  the 
vaginal  secretion  of  the  mare  did  not  exhibit  this  antimicrobial 
property  in  vitro,  but  in  the  other  animals  experimented  upon  it 
was  found  impossible  to  make  similar  experiments,  the  quantity  of 
mucus  being  too  small.  In  woman  the  acidity  of  the  surface  of  the 
mucous  membrane  of  the  vulva  and  of  the  vagina,  so  frequently 
present,  may  play  a  certain  part  in  the  protective  action  against  those 
bacteria  which  cannot  tolerate  the  acid  medium,  but  the  animals 
studied  by  Cahanescu,  even  female  dogs,  do  not  possess  this  advan- 
tage, their  mucous  membranes  usually  having  an  alkaline  reaction. 

In  the  urinary  channels  this  acid  reaction  also  plays  a  part,  as  one 
of  the  defensive  agencies  against  the  penetration  of  bacteria.  This 
may  also  be  effective  in  man  and  other  animals  that  have  an  acid  urine. 
In  many  other  animals,  however,  where  the  urine  is  alkaline  micro- 
organisms do  not  pass  into  the  deeper  parts  of  the  urinary  organ 
under  normal  conditions.  Here  it  is  to  the  outflow  of  the  urine  that 
the  bladder  owes  its  immunity  against  pathogenic  micro-organisms 
and  saprophytes.  When  we  connect  two  flasks  containing  sterilised 
broth  in  such  a  way  that  the  fluid  flows  slowly  from  one  of  them  into 
the  other,  the  former  never  becomes  contaminated  by  the  micro- 
organisms which  are  present  in  the  latter,  in  which  latter  the  broth 
is  soon  transformed  iuto  a  puree  of  bacteria,  whilst  in  the  former 
the  broth  remains  unaffected  and  aseptic.  This  purely  mechanical 
factor  has  been  well  brought  out  by  Preobrajensky1  as  the  result 
of  work  carried  out  in  Duclaux's  laboratory.  The  sterility  of  the 
normal  urinary  bladder  must  be  attributed  to  a  similar  cause.  When 
the  urine  begins  to  stagnate  in  the  bladder  it  very  readily  becomes 
contaminated. 

Since  the  acceptance  of  the  view  that  the  suprarenal  capsules 
serve  to  neutralise  the  effect  of  certain  toxic  substances  elaborated 
1  Ann.  de  Vlnst.  Pasteur,  Paris,  1897,  t.  xi,  p.  699. 


432  Chapter  XIII 

in  the  body,  there  has  been  an  inclination  to  assume  that  these 
organs  might  also  fulfil  an  antitoxic  role  against  microbial  poisons. 
The  hypothesis  was  advanced  that  this  function  might  be  shared 
by  the  suprarenal  capsules  with  the  thyroid  gland  and  with  certain 
other  problematical  organs.  We  have  already  stated  (Chapter  v) 
that  the  suprarenal  capsules,  in  some  experiments  where  spermo- 
toxin  was  injected  into  rabbits,  exhibited  a  certain  antispermotoxic 
[453]  power.  But,  up  to  the  present,  no  exact  fact  has  been  observed  that 
would  favour  the  idea  of  an  antitoxic  action  of  the  above-mentioned 
organs  against  bacterial  toxins.  Roux  and  Vaillard1,  in  their  great 
work  on  tetanus,  have  made  experiments  in  this  direction,  but  their 
results  did  not  justify  them  in  giving  a  positive  answer  to  the 
question. 

Nature  does  not  make  use  of  antiseptics  to  protect  the  skin  and 
the  mucous  membrane.  The  fluids  which  moisten  the  surface  of  the 
mouth  and  of  other  mucous  membranes  are  not  microbicidal,  or 
are  so  to  a  very  slight  degree,  and  then  rather  of  an  exceptional 
nature.  Nature  rids  the  mucous  membranes  and  the  skin  of  a  large 
number  of  micro-organisms,  eliminating  them  by  epithelial  desquama- 
tion,  and  expelling  them  along  with  fluid  secretions  and  excretions. 
Nature,  like  the  doctors  of  the  present  day  who  replace  antisepsis  of 
the  mouth,  intestine,  and  other  organs  by  washing  with  pure  physio- 
logical saline  solution,  has  chosen  this  mechanical  method.  She 
avails  herself  of  the  help  of  inoffensive  micro-organisms  to  prevent 
pathogenic  micro-organisms  from  taking  up  their  abode  in  these 
positions,  and  she  is  constantly  sending  to  all  the  mucous  membranes 
and  the  skin  an  army  of  mobile  phagocytes  which  explore  the  ground 
and  rid  it  of  micro-organisms.  When  these  begin  to  get  more 
numerous  the  phagocytic  reaction  becomes  more  intense.  A  struggle 
takes  place  between  the  two  living  elements — phagocytes  and  micro- 
organisms. In  those  cases  where  the  animal  remains  unaffected  the 
former  gain  the  upper  hand. 

1  Ann.  de  I'Inst.  Pasteur,  Paris,  1893,  t.  vu,  p.  65. 


CHAPTER  XIV  [454] 

IMMUNITY  ACQUIRED  BY  NATURAL  MEANS 

Immunity  acquired  after  recovery  from  infective  diseases. — Immunity  acquired  in 
malaria. — Humoral  properties  of  convalescents  from  typhoid  fever. — Preventive 
power  of  the  blood  of  persons  who  have  recovered  from  Asiatic  cholera, — 
Antitoxic  power  of  the  blood  of  persons  who  have  recovered  from  diphtheria. 

Immunity  acquired  by  heredity. — Absence  of  hereditary  immunity  properly  so-called. 
— Immunity  conferred  by  the  maternal  blood  and  by  the  yolk. 

Immunity  conferred  by  the  milk  of  the  mother. 

IT  has  long  been  known  that  an  attack  of  one  of  many  of  the  in- 
fective diseases  brings  about  a  refractory  condition  of  the  organism 
against  that  disease,  a  condition  which  persists  for  many  years,  and 
may  even  endure  for  life.  Even  before  the  microbiological  era  of 
medical  science  had  arrived  it  had  been  fully  established  that  a 
person  who  had  recovered  from  small-pox  might  come  in  contact 
with  and  nurse  small-pox  patients  without  risk  of  contracting  a 
second  attack  of  the  disease.  The  same  thing  has  been  observed 
purely  empirically  in  several  other  infective  diseases,  such  as 
whooping-cough,  typhoid  fever,  scarlatina,  mumps,  etc.  On  the 
other  hand  it  has  been  shown  that  certain  infective  diseases,  such 
as  fibriuous  pneumonia,  erysipelas,  recurrent  fever,  and  influenza, 
do  not  leave  behind  them  the  slightest  trace  of  an  immunity.  It 
has  often  been  observed,  indeed,  that  after  a  first  attack  of  any  of 
these  diseases  there  is  a  marked  susceptibility  to  a  second  attack. 
Between  these  two  extremes  come  the  infections  which  are  followed 
merely  by  a  refractory  condition  of  shorter  duration  than  that  which 
follows  the  diseases  of  the  first  group.  The  first  of  this  intermediate 
group  is  measles,  which  gives  rise  to  a  relatively  long  immunity,  then 
come  in  order  bubonic  plague,  anthrax,  cholera,  etc. 

It  should  be  stated  that  the  first  attack  of  any  of  the  infective 
diseases  causes  modifications  more  or  less  permanent  in  the  organism, 

B.  28 


434  Chapter  XIV 

and  is  always  followed  by  immunity.  Even  in  erysipelas,  a  disease 
[455]  where  the  relapses  are  so  frequent  that  certain  individuals  are,  so  to 
speak,  predestined  to  re-acquire  it  at  short  intervals,  an  immunity  is 
produced,  but  a  very  transient  one.  Since  the  discovery  of  the  strepto- 
coccus of  erysipelas  by  Fehleisen1,  this  observer,  and  several  other 
investigators,  have  inoculated  it  into  persons  affected  with  malignant 
tumours.  In  the  course  of  a  series  of  experimental  cases  of  treat- 
ment it  was  noted  on  several  occasions  that  after  a  first  inoculation, 
followed  by  typical  erysipelas,  a  period  of  immunity  was  developed, 
during  which  the  introduction  of  the  streptococcus  produced  no 
result.  It  has  also  been  observed  that  recurrent  fever,  when 
inoculated  into  monkeys,  sets  up  a  very  transient  but  real  re- 
fractory condition.  In  fibrinous  pneumonia,  also,  the  relapses  are 
generally  separated  by  periods  of  immunity,  of  longer  or  shorter 
duration. 

It  was  generally  supposed  that  an  attack  of  malarial  fever  was 
not  only  not  followed  by  any  immunity,  but  that  a  first  attack 
predisposed  the  organism  to  a  second.  Facts  of  this  kind  have 
often  been  observed  and  cannot  now  be  questioned.  Nevertheless, 
an  acquired  immunity  against  malaria  is  developed  under  certain 
conditions.  During  his  travels  in  New  Guinea,  Koch2  found  that  in 
certain  regions  whilst  most  children  below  ten  years  of  age  are 
attacked  by  malaria,  and  Laveran's  parasite  can  be  demonstrated  in 
their  blood,  older  children  and  adults  are  completely  immune  from 
this  infection.  Koch  is  convinced  that  in  this  instance  we  have  an 
example  of  immunity  acquired  by  natural  means  as  the  result  of  an 
attack  of  malaria  at  the  younger  age.  This  great  observer  bases  his 
conclusion  on  the  fact  that  unattacked  adults,  coming  from  districts 
where  the  children  contain  the  parasite,  do  not  contract  malaria  when 
they  migrate  to  other  malarial  regions,  whilst  natives  coming  into 
these  same  regions  from  districts  where  malaria  does  not  exist  are 
soon  attacked.  Max  Glogner3  has  attempted  to  explain  these  facts 
established  by  Koch,  on  the  assumption  that  the  unaffected  adults 
simply  benefit  by  their  natural  immunity  and  that  we  have  here 
a  kind  of  selection  :  the  adults  who  are  susceptible  ,to  malaria  die  as 
the  result  of  this  disease,  whilst  others,  naturally  refractory,  resist  and 
[456]  show  themselves  incapable  of  contracting  the  disease  even  in  other 

1  "Die  Etiologie  des  Erysipels,"  Berlin,  1883. 

2  Deutsche  med.  Wchnschr.,  Leipzig,  1900,  SS.  781,  801. 

3  Vir chow's  Archiv,  1900,  Bd.  CLXII,  S.  222. 


Immunity  acquired  by  natural  means  435 

malarial  regions.  Glogner  in  support  of  his  view  cites  the  case  of  the 
children  of  the  orphanage  at  Samarang  (Java),  who  for  many  years 
are  subject  to  relapses  and  to  malarial  re-infections  and  are  incapable 
of  acquiring  the  slightest  immunity.  According  to  Koch,  Glogner's 
example  cannot  be  compared  with  that  of  the  children  of  New  Guinea. 
In  the  former  case,  the  natural  course  of  the  disease  is  interrupted  by 
treatment  with  quinine,  which  must  prevent  immunity  being  set  up  ; 
whilst,  in  the  latter,  the  children  are  abandoned  to  their  fate,  and, 
receiving  no  treatment,  slowly  acquire  a  true  immunity.  It  is 
evident  that  this  acquired  immunity  in  malaria  is  a  complex  pheno- 
menon on  which  fresh  researches  must  be  made ;  but  it  cannot  be 
questioned  that,  under  certain  conditions,  it  comes  under  the  general 
rule  and  can  be  naturally  acquired. 

This  general  rule  is  that,  in  infective  diseases,  immunity  is 
usually  developed  after  a  first  attack.  The  acquired  refractory 
condition  is  of  very  long  duration  in  certain  cases,  but  very 
transitory  in  others.  To  the  discovery  of  the  vaccination  by  at- 
tenuated micro-organisms,  made  by  Pasteur  and  his  collaborators, 
the  objection  was  often  made  that  many  diseases,  such  as  anthrax, 
might  relapse.  This  is  undoubtedly  the  case;  the  anthrax  bacillus 
may  attack  the  same  individual  several  times;  nevertheless  the 
acquired  immunity  against  this  disease  is  very  real,  though  the 
refractory  condition  lasts  for  one  or  a  few  years  only,  instead  of 
persisting  for  a  very  much  longer  period,  as  in  the  case  of  typhoid 
fever,  mumps,  and  small-pox.  Bearing  in  mind  the  possibility  of  a 
relapse  in  the  case  of  these  infective  maladies,  attempts  at  artificial 
vaccination  should  never  be  relinquished. 

Among  the  examples  of  immunity  acquired  by  natural  means 
must  be  cited  that  of  syphilis,  a  very  special  case.  It  has  long  been 
known  and  demonstrated  by  numerous  experiments  on  man,  that 
individuals  who  have  presented  the  primary  symptoms  of  syphilis 
contract  a  marked  immunity  against  a  new  infection.  The  syphilitic 
chancre  does  not  relapse,  and  yet  this  very  manifest  and  persistent 
immunity  does  not  prevent  the  individual,  immune  against  re-infec- 
tion, from  continuing  to  be  ill  and  of  being  the  field  for  the  later 
syphilitic  phenomena.  This  special  refractory  condition  has  done 
great  service  in  establishing  the  etiology  of  certain  diseases  which  we  [457] 
were  justified  in  suspecting  to  be  of  syphilitic  origin.  Many  clinical 
observers  have  accepted  this  origin  for  general  progressive  paralysis. 
Others  deny  any  causal  relation  between  the  two  diseases.  Krafft- 

i'S— 2 


436  Chapter  XIV 

Ebing1  has  resolved  this  question  by  the  application  of  the  law  of 
acquired  syphilitic  immunity.  The  inoculation  of  the  syphilitic  virus 
into  ten  persons  attacked  by  general  paralysis  was  followed  by  no 
chancre  at  the  seat  of  inoculation  and  by  no  other  primary  or 
secondary  symptom  of  syphilis.  The  patients  with  general  paralysis 
present  a  real  immunity  against  these  symptoms ;  consequently 
general  paralysis  is  a  tardy  manifestation  of  syphilis. 

The  acquired  immunity  against  re-inoculation  by  the  syphilitic 
virus  is  established  immediately  after  the  end  of  the  period  of  incuba- 
tion of  the  first  infection,  and  is  of  lifelong  duration2.  Besides  this 
very  special  and,  so  to  speak,  partial  immunity,  there  exists  in  syphilis 
a  second  form  of  acquired  immunity  which  is  of  a  more  general 
nature.  According  to  the  law  known  as  the  law  of  Baumes-Colles, 
the  mother  who  suckles  her  infant,  hereditarily  infected  with  syphilis 
through  the  father  only,  enjoys  a  real  anti-syphilitic  immunity. 

In  tuberculosis  the  few  facts  of  acquired  immunity  that  have 
been  observed  present  a  certain  analogy  with  those  bearing  on  im- 
munity in  syphilis.  A  large  number  of  well-observed  facts  demon- 
strate that  a  person  who  has  suffered  from  scrofula  or  has  manifest 
symptoms  of  tuberculosis  properly  so  called,  cannot  count  upon  an 
immunity  against  pulmonary  phthisis.  It  might,  then,  be  supposed 
that  no  acquired  refractory  condition  exists  in  connection  with  this 
disease.  Koch3  has  clearly  demonstrated,  however,  that  tuberculous 
guinea-pigs,  into  which  the  bacilli  of  tuberculosis  have  been  intro- 
duced subcutaneously,  react  against  these  bacilli  in  a  very  special 
manner.  The  presence  of  these  micro-organisms  immediately  sets  up 
an  active  inflammatory  process  at  the  point  of  inoculation  ;  this  brings 
about  the  expulsion  of  the  bacilli  with  the  exudation  ;  a  voluminous 
slough  is  developed,  which,  when  shed,  carries  with  it  a  large  number 
of  bacilli,  a  process  followed  neither  by  the  formation  of  a  permanent 
ulcer  nor  by  hypertrophy  of  the  neighbouring  glands.  As  in  syphilis, 
the  animal  has  acquired  immunity  against  re-infection  by  the  tuber- 
[458]  culous  virus,  which,  however,  in  no  way  prevents  the  first  inoculation 
from  becoming  generalised  and  setting  up  a  fatal  tuberculosis  of 
almost  all  the  organs.  Koch's  observations,  which  have  served  as  the 
basis  of  his  researches  on  tuberculin,  have  been  confirmed  by  other 

1  Address  given  at  the  Xllth  International  Congress  of  Medicine  at  Moscow, 
1897. 

2  See  Hudalo,  Ann.  de  dermat.  et  de  syph.,  Paris,  1891,  t.  n,  pp.  353,  470. 

3  Deutsche  med.  Wchnschr.,  Leipzig,  1891,  S.  101. 


Immunity  acquired  by  natural  means  437 

investigators.     The  reaction  of  the  tuberculous  organism  against  re- 
infection has  received  the  name  of  "Koch's  phenomenon." 

Clinical  medicine  has  afforded  many  data  of  the  highest  import- 
ance bearing  on  the  establishment  of  an  acquired  immunity  in  many 
infective  diseases ;  but  a  scientific  study  of  the  mechanism  of  this 
immunity  could  only  be  founded  on  the  result  of  microbiological 
researches  obtained  during  the  recent  period  of  scientific  activity. 
The  general  conclusion  to  be  drawn  from  these  researches  is  that  the 
immunity,  acquired  by  natural  means,  is  very  analogous  to  that  which 
is  obtained  artificially  by  vaccination  by  the  various  methods  already 
mentioned.  The  phenomena  observed  in  animals  inoculated  with  the 
various  known  vaccines  present  a  great  resemblance  to  those  that 
obtain  during  recovery  from  a  disease  contracted  under  natural  con- 
ditions. To  support  this  thesis  it  would  be  necessary  for  us  to  survey 
the  mechanism  of  healing,  which  would  carry  us  too  far  afield,  the 
subject  being  far  too  vast  to  be  summarised  here.  We  must,  then, 
content  ourselves  with  a  few  remarks  inserted  for  the  instruction 
of  the  reader  on  this  subject. 

Those  diseases  against  which  no  remedy  exists  are  most  suitable 
for  furnishing  us  with  important  information  on  immunity  acquired 
by  natural  means.  We  have  already  seen  in  the  case  of  malaria  to 
what  point  therapeutic  treatment  can  modify  the  natural  course  of 
the  phenomena.  For  this  reason  it  will  be  useful  to  consider  first  the 
immunity  acquired  as  the  result  of  a  first  attack  of  typhoid  fever. 
The  immunity  which  develops  in  this  example  is  both  marked  and 
persistent ;  the  therapeutic  intervention  which  might  disturb  the 
natural  phenomena  is  nil. 

As  yet  we  do  not  know  the  mechanism  of  healing  in  typhoid  fever. 
This  disease  affecting  the  human  species  exclusively  (the  experi- 
mental peritonitis  of  animals,  set  up  by  the  typhoid  coccobacillus,  is 
distinguished  by  very  marked  differences),  it  is  very  difficult  to  find  a 
means  of  studying  it  at  all  satisfactorily  during  the  phase  of  recovery. 
Even  in  default  of  this  knowledge,  however,  it  is  possible  to  gather  [459] 
some  idea  as  to  the  changes  which  the  blood  plasma  undergoes,  not 
only  during  the  course  of  an  attack  of  typhoid  fever,  but  also  during 
and  after  convalescence. 

Some  time  ago  Chantemesse  and  Widal1  observed  that  the  blood 
serum  of  persons  attacked  by  typhoid  fever  acquires  the  property  of 
inhibiting  the  experimental  peritonitis  set  up  by  the  typhoid  cocco- 
1  Ann.  de  FInst.  Pasteur,  Paris,  1892,  t.  vi,  p.  773. 


438  Chapter  XIV 

bacillus  in  laboratory  animals.  The  blood  of  the  patient  becomes 
"  preventive."  Against  this  conclusion  the  objection  has  been  raised 
that  in  the  large  doses  of  serum  employed  by  the  above  observers 
a  protective  effect  can  be  obtained,  even  when  using  the  blood  of 
normal  men,  i.e.  neither  suffering  from  typhoid  fever,  nor  having 
recovered  from  this  disease.  Later  researches,  however,  have  con- 
firmed the  observation  made  by  Chan  tern  esse  and  Widal.  It  is  no 
doubt  true  that  the  injection  of  half  a  cubic  centimetre  of  normal 
human  serum  into  the  peritoneal  cavity  of  an  untreated  guinea-pig  is 
often  sufficient  to  render  it  refractory  to  a  dose  of  typhoid  cocco- 
bacilli  fatal  to  the  control  animal.  We  have  an  ordinary  protective 
action,  such  as  described  in  Chapter  x.  The  blood  of  typhoid 
patients  is,  however,  capable  of  protecting  normal  animals,  in  doses 
which  exhibit  not  the  slightest  protective  action  if  normal  blood  be 
used. 

The  protective  power  of  the  blood  serum  of  convalescents  has  been 
studied  very  carefully  by  Pfeiffer  and  Kolle1.  In  certain  individuals 
very  small  quantities  (0*001  c.c.)  of  this  fluid  were  quite  sufficient  to 
confer  on  guinea-pigs  an  immunity  against  fatal  typhoid  peritonitis. 
This  power  was  at  its  maximum  only  during  the  first  weeks  of  con- 
valescence. In  one  case,  in  which  these  observers  were  able  to  study 
the  properties  of  the  blood  on  two  separate  occasions,  they  found 
that  two  months  after  the  first  examination  there  had  been  a  marked 
falling  off  in  the  acquired  protective  power.  In  a  second  case,  where 
the  blood  was  examined  a  year  after  the  patient  had  recovered  from 
a  grave  attack  of  typhoid  fever,  they  found  only  feeble  indications 
of  this  specific  protective  property.  "  Everything  seems  to  indicate," 
conclude  Pfeiffer  and  Kolle,  "  that  the  protective  typhoid  substances 
were  rapidly  eliminated  by  the  blood  stream.  If  further  researches 
should  confirm  these  results,  as  yet  few  in  number,  we  might  conclude 
therefrom  that  the  immunity  which,  after  an  attack  of  typhoid  fever, 
[460]  persists  for  years,  frequently  even  for  the  rest  of  life,  would  be  in- 
dependent of  the  amount  of  ready-prepared  protective  substances 
in  the  blood"  (l.c.  p.  218).  The  facts  upon  which  this  conclusion  is 
based  confirm  the  general  thesis  that  even  acquired  immunity  is  in 
no  way  the  function  of  any  humoral  property. 

We  know  that  in  the  protective  serums  there  is  constantly  found 
the  specific  fixative  (the  sensibilising  substance  of  Bordet,  the  inter- 
mediary body  or  amboceptor  of  Ehrlich).    It  was,  therefore,  quite 
1  Ztschr.f.  Hyg.,  Leipzig,  1896,  Bd.  xxi,  S.  213. 


Immunity  acquired  by  natural  means  439 

natural  that  this  substance  should  be  sought  in  the  blood  of  patients 
who  were  suffering,  or  had  recovered,  from  typhoid  fever.  Bordet  and 
Gengou1  easily  demonstrated,  by  the  method  described  in  Chapter  ix, 
the  existence  of  typhofixative  in  the  blood  serum  of  two  individuals 
convalescing  from  this  disease. 

Widal  and  Le  Sourd2  extended  this  discovery  to  the  blood  taken 
during  the  course  of  the  disease  from  typhoid  fever  patients.  The 
ten  cases  studied  by  them  all  gave  a  positive  result,  whilst  all  the 
samples  of  blood  from  persons  suffering  from  various  other  diseases 
possessed  no  typhofixative.  As  yet  we  do  not  know  whether  this 
substance  persists  for  any  length  of  time  after  recovery  or  not.  In 
this  respect  we  have  much  more  information  concerning  another 
humoral  property  of  typhoid  patients, — specific  agglutination.  Guided 
by  the  fact  that,  even  during  the  course  of  the  disease,  the  blood  of 
persons  suffering  from  typhoid  fever  acquires  protective  properties, 
Widal  sought  to  find  out  whether  the  agglutinative  power  of  the 
fluids  of  the  body  appears  equally  early.  We  know  that  his  studies 
gave  a  positive  answer,  and  that  the  blood  of  typhoid  patients  may 
have  agglutinative  properties  from  the  first  day  of  the  disease.  This 
fact  was  made  use  of  by  Widal  to  establish  the  serum  diagnosis  of 
typhoid  fever,  a  method  now  generally  used  in  clinical  medicine. 
The  question  which  most  interests  us  at  this  moment  is  whether  this 
acquired  agglutinative  property  persists  for  any  length  of  time  after 
the  recovery  of  the  patient,  and  whether  it  can  be  employed  as  the 
measure  of  immunity  obtained. 

In  certain  cases  the  serum  was  found  to  be  fairly  strongly  aggluti- 
native for  a  considerable  period  after  recovery  had  taken  place.  But 
these  cases  are  rare,  and  the  agglutinative  power,  like  the  protective 
power  of  the  blood,  usually  begins  to  decrease  very  soon  after 
recovery.  Bensaude3  observed  that  the  former  disappeared  between  [461] 
the  10th  and  95th  day  of  apyrexia,  Widal  and  Sicard4  have  noted  in 
certain  of  their  cases  the  complete  disappearance  of  the  agglutinative 
power  of  the  blood,  which  took  place  in  one  case  on  the  18th,  in 
another  on  the  24th  day  of  defervescence.  In  many  convalescents, 
fifteen  to  thirty  days  after  the  commencement  of  apyrexia,  the  agglu- 
tinative power  begins  to  be  attenuated. 

1  Ann.  de  VInst.  Pasteur,  Paris,  1901,  t.  xv,  p.  289. 

55  Bull,  et  mem.  Soc.  med.  d.  hop.  de  Paris,  1901,  20  juin,  p.  624. 

3  "Le  phenomene  de  1'agglutination  des  microbes,"  Paris,  1897,  p.  76. 

4  Presse  med.,  Paris,  1896,  No.  83. 


440  Chapter  XIV 

Previous  to  these  researches  on  the  protective  and  agglutinative 
properties,  Stern1  had  already  put  the  question :  May  we  not  draw 
gome  general  conclusion  as  to  the  bactericidal  power  of  the  blood 
serum  of  convalescents  from  typhoid  fever?  He  found  that  the 
typhoid  coccobacilli  did  not  thrive  so  well  in  the  blood  serum  of 
persons  in  good  health  as  in  that  of  convalescents,  in  which  they  give 
abundant  cultures.  Widal  and  Sicard  (Lc.)  subjected  this  question 
to  a  fresh  examination,  and  showed  that  in  this  respect  there 
exists  no  constant  or  marked  difference.  Thus,  in  ten  samples  of 
serums  from  individuals  who  had  never  been  under  the  influence  of 
the  typhoid  infection,  four  were  found  to  be  bactericidal  for  the 
typhoid  coccobacillus.  In  twelve  other  samples,  drawn  from  con- 
valescents from  typhoid  fever,  five  exhibited  a  bactericidal  power 
against  the  same  micro-organism. 

All  the  researches  made  on  acquired  immunity  after  recovery  from 
typhoid  fever  demonstrate  clearly  that,  in  this  case,  it  is  impossible  to 
attribute  it  to  humoral  modifications,  which  are  usually  more  transi- 
tory than  the  immunity. 

The  immunity  which  follows  an  attack  of  cholera  is  far  from  being 
either  as  powerful  or  as  prolonged  as  that  which  follows  typhoid  fever. 
Certain  individuals  have  contracted  cholera  twice  during  the  same 
epidemic,  but  such  cases  are  exceptional,  whilst  acquired  immunity, 
temporary  at  least,  may  be  looked  upon  as  the  general  rule.  Many 
points  in  the  pathogenesis  of  intestinal  cholera  are  still  obscure  ; 
nevertheless  we  are  justified  in  affirming  that  this  disease  is  a  real 
intoxication  by  the  cholera  poison  manufactured,  in  the  small  intestine 
of  man,  by  Koch's  vibrios.  The  action  of  the  vibrionic  toxin  is 
sufficient  to  set  up  a  grave  and  often  fatal  attack  of  cholera  ;  but  in 
the  majority  of  cases  a  secondary  infection  by  the  vibrio  which  pene- 
[462]trates  into  the  intestinal  wall,  denuded  of  its  epithelial  layer,  is 
associated  with  the  action  of  the  poison.  Sometimes  this  micro- 
organism becomes  generalised  in  the  animal,  and  is  found  in  the 
blood  and  in  many  of  the  organs. 

The  facts  I  have  here  briefly  summarised  may  be  utilised  to  explain 
certain  characters  which  are  found  in  the  fluids  of  individuals  who  have 
recovered  from  an  attack  of  cholera.  Soon  after  the  discovery  of  the 
tetanus  and  diphtheria  antitoxins,  and  almost  immediately  after  the 
demonstration  of  the  protective  power  of  the  blood,  taking  advantage 
of  the  epidemic  of  Asiatic  cholera,  which  developed  in  Europe  from 
1  Deutsche  med.  Wchnschr.,  Leipzig,  1892,  S.  827. 


Immunity  acquired  by  natural  means  441 

1892,  the  new  data  began  to  be  applied  to  that  disease.  We  have 
already  referred  in  a  preceding  chapter  to  the  fact  that  the  blood 
serum  or  the  blood  of  those  in  good  health  and  who  have  never  had 
Asiatic  cholera,  is  capable  of  preventing  cholera  peritonitis  in  the 
guinea-pig  inoculated  with  Koch's  vibrios.  In  order  to  obtain  this 
protective  action,  the  injection  of  a  pretty  large  dose,  about  half  a  c.c., 
is  necessary.  This  property  is  in  no  sense  specific,  for  the  same 
blood,  injected  in  the  same  doses  into  guinea-pigs,  will  protect 
them  not  only  against  this  vibrio,  but  also,  and  indifferently,  against 
many  other  bacteria,  such  as  the  typhoid  coccobacillus,  the  Bacillus 
coU.  etc. 

The  blood  or  blood  serum,  coming  from  those  who  have  recovered 
from  Asiatic  cholera,  may,  on  the  other  hand,  acquire  a  specific 
protective  power.  It  will,  indeed,  prevent  infections  by  other  micro- 
organisms ;  but,  to  obtain  this  effect,  it  is  necessary  to  inject  the  same 
quantities  of  it  as  of  the  blood  coming  from  normal  individuals.  On 
the  other  hand,  when  we  wish  to  prevent  cholera  peritonitis  in  the 
guinea-pig,  we  need  introduce  minute  doses  only  of  the  serum  of 
persons  who  have  recovered  from  an  attack  of  cholera,  Lazarus1  was 
the  first  to  make  this  interesting  observation.  In  three  cases  of 
cholera  studied  by  him,  the  serum  withdrawn  some  time  after  recovery 
exhibited  an  extraordinary  protective  power :  a  decimilligramme  of 
the  blood  serum  of  these  patients  was  quite  sufficient  to  prevent  the 
death  of  a  guinea-pig  inoculated  intraperitoneally  with  the  cholera 
vibrio.  Soon  after,  G.  Klemperer2  made  a  similar  observation  in  two 
other  cases  that  had  recovered,  but  the  blood,  in  his  convalescents, 
was  much  less  active  than  was  that  in  the  cases  cited  by  Lazarus. 

Issaeff 3,  working  in  Koch's  Institute  in  Berlin,  examined  the  blood 
of  several  persons  who  had  recovered  from  cholera,  and  found  that  [463] 
the  serum  had  always  acquired  a  specific  protective  property ;  this 
property  never  developed  before  the  third  week  from  the  commence- 
ment of  the  disease,  and  had  completely  disappeared  as  early  as 
three  months  after  this  period.  Several  examples  studied  by 
A.  Wassermann4  and  Sobernheim5  fully  corroborate  this  conclusion. 
Our  own  researches6  on  twenty-four  cases  indicate  a  very  great 

1  Berl  klin.  Wchnschr.,  1892,  S.  1072;  1893,  S.  1241. 
*  Berl  klin.  Wchnschr.,  1892,  S.  1267. 

3  Ztschr.f.  Hyg.,  Leipzig,  1894,  Bd.  xvi,  S.  308. 

4  Ztschr.f.  Hyg.,  Leipzig,  1893,  Bd.  xiv,  S.  42. 

5  Hyg.  Rundsch.,  Berlin,  1895,  S.  145. 

6  Ann.  de  I'Inst.  Pasteur,  Paris,  1893,  t.  vn,  p.  417. 


442  Chapter  XIV 

variability  in  the  protective  power  of  the  blood  of  persons  who  had 
recovered  from  cholera.  We  were  able  to  demonstrate  its  presence 
in  rather  more  than  58  per  cent,  of  these  cases.  Sometimes  this 
power  was  almost  as  marked  as  in  the  example  given  by  Lazarus, 
whilst  in  others  it  was  very  feeble,  often  even  nil.  We  were  unable 
to  demonstrate  any  relation  between  the  gravity  of  the  disease  and 
the  strength  of  the  protective  power  of  the  blood.  Thus,  in  a  mode- 
rately severe  case  of  cholera,  a  very  small  quantity  of  serum  (O'OOl  c.c.) 
was  sufficient  to  protect  the  guinea-pig  from  fatal  cholera  peritonitis, 
whilst  in  another,  an  extraordinarily  grave  case,  even  a  quantity  of 
2  c.c.  was  incapable  of  producing  the  same  effect.  In  these  two  cases 
the  blood  had  been  withdrawn  at  the  corresponding  period  after  the 
commencement  of  the  disease  (seventy-third  and  seventy-fifth  days). 
Sobernheim  (I.e.)  found  the  protective  power  of  the  serum  most 
marked  in  a  person  who  had  cholera  vibrios  in  his  normal  dejecta, 
but  who  was  always  in  good  health  and  was  only  examined  because 
he  was  living  with  cholera  patients. 

All  these  observations  point  to  the  fact  that  neither  recovery  from, 
nor  immunity  against,  cholera  can  be  regarded  as  a  consequence  of 
the  protective  power  of  the  blood.  This  power  does  not  manifest 
itself  until  some  time  after  complete  recovery  has  taken  place,  and 
then  disappears  too  soon,  that  is  to  say  at  a  moment  when  acquired 
immunity  ought  still  to  be  maintained.  On  the  other  hand,  the 
irregularity  in  the  protective  power  of  the  blood  indicates  that  this 
humoral  property  is  something  secondary.  Since  Asiatic  cholera  is  an 
intoxication  by  the  cholera  toxin,  we  can  readily  understand  that  the 
protective  power,  resulting  from  the  invasion  of  the  living  parts  of 
the  organism  by  the  vibrios,  should  here  play  a  part  of  little  import- 
ance. We  know  already  that  this  power  is  due  to  the  presence  of 
substances  manufactured  by  phagocytic  elements,  placed  in  contact 
[464]  with  vibrios.  In  the  experimental  infection  of  rabbits  by  the  cholera 
vibrio,  as  demonstrated  by  Pfeiffer  and  Marx,  the  cells  of  the  spleen, 
of  the  lymphatic  glands,  and  of  the  bone-marrow,  produce  the  pro- 
tective substances.  We  have  no  idea  of  the  source  of  these  substances 
in  Asiatic  cholera  in  man. 

Asiatic  cholera,  being  an  example  of  intoxication  of  intestinal 
origin,  it  might  be  supposed  that  the  antitoxic  power  of  the  body 
fluids  should  be  specially  manifested  after  recovery  has  taken  place. 
On  this  point  our  knowledge  is  as  yet  very  imperfect,  because  it  was 
not  until  after  the  end  of  the  last  epidemic  of  cholera  that  we  learnt 


Tnununity  acquired  by  natural  means  443 

how  to  prepare  the  toxin.  In  a  case  of  cholera  (M.S.),  contracted 
in  our  laboratory,  the  blood  serum  was  examined  to  ascertain  its 
protective  power  and  its  antitoxic  activity.  This  fluid,  withdrawn 
more  than  three  weeks  after  the  commencement  of  the  disease,  was 
found  to  be  protective  only  in  a  large  dose  (0*5  c.c.),  in  which  dose 
even  the  serum  of  normal  persons  is  capable  of  producing  the  same 
effect.  It  was  found  in  an  experiment  with  suckling  rabbits  that  the 
antitoxic  property  of  the  blood  serum  of  M.  S.  was  nil.  It  did  not 
prevent  these  rabbits  from  dying  of  intestinal  cholera  after  the 
absorption  of  the  vibrios,  in  spite  of  a  dose  of  three  c.c.  of  serum 
injected  some  time  previously. 

This  experiment,  unique  up  to  the  present,  is,  of  course,  insufficient 
to  enable  us  to  affirm  that  recovery  from  Asiatic  cholera  may  take 
place  without  the  development  of  antitoxic  power  in  the  body  fluids. 
That  this  is  so  is,  nevertheless,  probable.  In  other  intoxications  of 
microbial  origin,  certain  data  have  been  collected  which  point  to  the 
same  conclusion.  Thus,  Knorr1  observed  that  the  blood  of  guinea- 
pigs  which  had  recovered  from  tetanus  did  not  exhibit  any  antitetanic 
power.  Vincenzi2  made  a  similar  observation  in  a  man  who  had 
recovered  from  tetanus. 

We  are  much  better  informed  as  to  the  antitoxic  property  of  the 
blood  of  persons  who  have  recovered  from  diphtheria.  Klemensiewicz 
and  Escherich3  have  studied  two  cases  of  diphtheria  in  which  the 
defibrinated  blood  withdrawn  some  time  after  recovery  was  found  to 
be  protective  for  the  guinea-pig  against  a  lethal  dose  of  diphtheria 
bacilli.  This  fact  has  been  confirmed  by  several  other  observers, 
especially  by  Abel4  and  Orlowski5,  the  latter  of  whom  made  his  [465] 
researches  under  the  direction  of  Escherich.  In  these  experi- 
ments the  antitoxic  power  of  the  blood  was  demonstrated  against 
diphtheria  toxin  employed  without  bacilli.  According  to  the  data 
collected  by  the  above  authors  the  antitoxic  property  of  the  body 
fluids  was  not  exhibited  during  the  early  days  of  convalescence,  but 
was  well  marked  in  the  second  week  after  recovery.  This  power  was 
maintained  for  a  short  time  only,  disappearing  in  a  few  months. 
Amongst  the  observations  collected  on  this  subject  the  most  inter- 

1  Munchen.  med.  Wchnschr.,  1898,  8.  363. 

2  Deutsche  med.  Wchnschr.,  Leipzig,  1898,  S.  247. 

8  Centralbl.f.  Bakteriol  u.  Parasitenk.,  Jena,  1893,  Bd.  xm,  S.  153. 

4  Deutsche  med.  Wchnschr.,  Leipzig,  1894,  SS.  899,  936. 

5  Deutsche  med.  Wchnschr. ,  Leipzig,  1895,  S.  400. 


444  Chapter  XIV 

esting  is  that  made  by  Escherich.  In  an  infant  examined  for  the 
first  time  whilst  it  was  still  in  good  health,  the  blood  was  incapable 
of  protecting  the  guinea-pig.  Some  time  after  this  negative  result 
had  been  obtained  the  child  was  attacked  by  a  mild  diphtheria,  which 
gave  rise  to  the  development  of  antitoxin,  for  its  blood  when  again 
examined  exhibited  a  very  high  antitoxic  power.  This  proves  most 
clearly  that  even  a  slight  attack  of  diphtheria  is  capable  of  producing 
antitoxic  power  in  the  body  fluids.  This  observation  may  be  utilised 
to  explain  the  frequency  of  the  presence  of  this  property  in  the  blood 
of  persons  in  good  health  who,  according  to  their  own  statements, 
have  never  had  diphtheria.  This  fact  has  been  established  by  the 
researches  of  A.  Wassermann1,  Abel  (l.c.),  and  Orlowski.  According 
to  the  last  observer,  the  blood  in  one-half  the  children  in  the  hospital 
at  Gratz  who  had  not  been  attacked  with  diphtheria  was  antitoxic 
against  the  diphtheria  toxin,  sometimes  even  to  a  higher  degree 
than  was  that  of  the  children  who  had  recovered  from  this  disease. 
Wassermann  has  demonstrated  that  in  adults  this  antidiphtheritic 
power  of  the  blood  is  even  more  frequent  than  in  children,  and  that  it 
increases  with  age.  Nevertheless,  these  persons  affirm  that  they  have 
never  had  an  attack  of  the  disease.  To  explain  this  very  paradoxical 
fact,  Wassermanu  asked  himself  whether  the  individuals  whose  blood 
was  antidiphtheritic  did  not  owe  this  property  to  the  action  of 
pseudo-diphtheria  bacilli.  Although  incapable  of  causing  the  disease, 
these  bacilli  might,  perhaps,  exert  a  certain  immunising  influence 
and  give  rise  to  the  production  of  an  antitoxin  active  against  true 
diphtheria  toxin.  Researches,  directed  to  the  clearing  up  of  this 
point,  have  not  led  Wassermann  to  reaffirm  his  suggestion.  It  must 
be  observed  that  the  varieties  of  these  pseudo-diphtheria  bacilli  are 
[466]  numerous,  and  that  some  of  them,  perhaps,  may  be  capable  of  fulfilling 
the  function  suggested  by  Wassermann.  On  the  other  hand,  it  is 
proved  that  the  specific  and  virulent  diphtheria  bacillus  may  be 
found  in  the  throat  of  persons  in  good  health  either  without  inducing 
diphtheria,  or  only  giving  rise  to  a  very  slight  form  of  disease  of  very 
short  duration.  We  must  bear  in  mind  that  in  persons  who  have 
not  had  typhoid  fever,  but  who  live  among  patients  suffering  from 
this  disease,  the  blood  may  be  very  agglutinative  (Foerster) ;  that  in 
others,  unattacked  by  cholera  but  containing  Koch's  vibrios  in  the 
intestine,  the  blood  may  acquire  a  high  specific  protective  power 
(Sobernheim).  It  is  probable  that  the  same  rule  applies  also  to  the 
1  Ztschr.f.  Hyg.,  Leipzig,  1895,  Bd.  xix,  S.  403. 


Immunity  acquired  by  natural  means  445 

case  of  diphtheria,  and  that,  consequently,  the  blood  of  persons  in 
good  health,  but  containing  the  diphtheria  bacillus  in  their  bodies, 
may  acquire  antitoxic  power. 

This  humoral  power,  once  developed,  may  even  be  transmitted 
from  the  mother  to  the  foetus  and  so  become  hereditary.  Abel  (l.c.) 
examined  the  blood  serum  of  four  adult  women,  taking  it  from 
the  placenta  after  parturition.  Each  time  it  was  found  to  be  dis- 
tinctly antitoxic  against  the  diphtheria  toxin.  Later,  Fischl  and 
AVunschheim1,  working  in  Chiari's  laboratory  in  Prague,  studied  the 
blood  of  new-born  children  from  the  same  point  of  view.  They 
showed  that  in  the  majority  of  cases  this  fluid  prevents  the  produc- 
tion of  a  fatal  disease  in  the  guinea-pig,  in  spite  of  the  injection  of 
several  lethal  doses  of  very  virulent  diphtheria  cultures.  The  blood 
of  new-born  children  is  equally  capable  of  neutralising  the  diphtheria 
toxin,  that  is  to  say,  of  protecting  animals  against  poisoning  by  this 
toxin.  The  above  observers  do  not  doubt  that  this  antitoxic  power 
comes  directly  from  the  maternal  blood  through  the  placental  circula- 
tion. This  fact  appears  to  throw  some  light  on  the  phenomena  of 
immunity  acquired  by  heredity. 

Until  quite  recently  we  have  had  very  vague  notions  as  to  the 
possibility  of  transmitting  to  descendants  the  immunity  contracted  as 
the  result  of  recovery  from  an  infective  disease  or  after  vaccination. 
It  has  long  been  known  that  natural  immunity  may  be  transmitted 
hereditarily.  Certain  families  or  certain  races  are  characterised  by 
a  special  insusceptibility  to  certain  infective  diseases.  It  must  even  [467] 
be  admitted  that  this  innate  immunity  has  been  transmitted  from 
generation  to  generation.  It  is  quite  otherwise  with  acquired  immu- 
nity. We  know  that  as  a  rule  the  characters  acquired  during  life 
are  not  transmitted  to  descendants ;  it  is  only  in  special  cases, 
in  the  very  lowest  organisms,  such  as  the  bacteria  and  their  allies, 
that  we  may  observe  the  conservation  of  certain  acquired  characters 
through  an  infinity  of  generations.  The  attenuation  of  bacteria  or 
the  absence  of  the  formation  of  spores,  once  acquired  under  special 
conditions,  may  thus  be  transmitted  to  their  descendants  who  develop 
and  live  under  normal  conditions. 

After  the  discovery  of  anthrax  vaccine  by  Pasteur,  Chamberland 

and  Roux,  and  an  attempt  had  been  made  to  vaccinate  large  flocks 

of  sheep,  it  was  an  easy  matter  to  investigate  whether  immunity 

acquired  by  the  parents  was  transmissible  to  their  offspring.    Several 

1  Prag.  med.  Wchnschr.,  1896. 


446  Chapter  XIV 

observers,  amongst  whom  I  may  specially  cite  Chauveau1,  Rossignol 
and  Cienkowski,  got  together  a  certain  number  of  data  bearing  on 
this  question.  These  data  showed  distinctly  that,  in  certain  cases, 
the  lambs  born  from  vaccinated  sheep  presented,  from  birth,  an  un- 
doubted resistance  to  the  anthrax  bacillus.  This  fact,  however,  was 
neither  constant  enough  nor  sufficiently  marked  to  enable  us  to  count 
upon  the  young  animals  being  in  a  refractory  condition,  and  thus 
avoid  having  to  submit  them  to  vaccination  by  the  two  Pasteur 
vaccines.  This  necessity  threw  into  the  background  the  researches 
on  the  hereditary  transmission  of  acquired  immunity.  It  was  only 
much  later  that  this  question  was  again  taken  up  on  a  purely 
theoretical  basis.  Ehrlich2,  to  whom  science  is  indebted  for  so  many 
works  of  the  highest  importance  upon  immunity,  again  took  the 
initiative  in  exact  and  minute  researches  upon  the  heredity  of 
immunity,  acquired  as  the  result  of  vaccination  against  toxins.  In 
this  relation  he  studied  the  immunity  of  the  descendants  of  animals 
immunised  against  phanerogamic  toxins,  such  as  ricin,  abrin  and 
robin,  and  later,  in  collaboration  with  Hiibener3,  that  of  the  offspring 
of  animals  vaccinated  against  tetanus  toxin.  Ehrlich  proved  very 
clearly  that  the  antitoxic  immunity  acquired  by  the  father  is  never 
[468]  transmitted  to  his  progeny.  This  fact  alone  is  quite  sufficient 
to  show  that  it  is  not  a  true  immunity  that  is  met  with  in  young 
animals  born  of  mothers  who  have  acquired  a  refractory  condition ; 
true  immunity  is  transmitted  by  the  sexual  elements,  the  sper- 
matozoon and  the  ovum.  Certain  observers,  Tizzoni4  and  his 
collaborators  Cattani  and  Centanni,  thought  they  could  overthrow 
the  rule  established  by  Ehrlich.  They  believed  that  the  male  rabbit, 
vaccinated  against  rabies,  was  capable  of  transmitting  its  immunity 
to  its  progeny.  Charrin  and  Gley5  expressed  the  same  opinion  as 
regards  animals  of  the  male  sex  vaccinated  against  experimental 
pyocyanic  disease.  But  the  very  precise  experiments  of  Wernicke6, 

1  Ann.  de  rimt.  Pasteur,  Paris,  1888,  t.  n,  p.  69. 

2  Ztschr.f.  Hyg.,  Leipzig,  1892,  Bd.  xn,  S.  183;  Brieger  u.  Ehrlich,  Deutsche 
med.  Wchnschr.,  1892,  8.  393. 

3  Ztschr.f.  Hyg.,  Leipzig,  1894,  Bd.  xvm,  S.  57. 

4  Centralbl.  f.  Bakteriol.  u.  Parasitenk.,  Jena,  1893,  Bd.  xni,  S.  81 ;  Deutsche 
med.  Wchnschr.,  Leipzig,  1892,  S.  394. 

5  Compt.  rend.  Acad.  d.  «?.,  Paris,  1893,  t.  cxvn,  p.  365  ;  Ren.  gen.  d.  sc.  pures  et 
appliq.,  Paris,  1896,  p.  1. 

6  Festschr.  z.  100-jahr.  Stiftungsf.  d.  med.  chir.  Friedr.   Wilhelms-Instituts, 
Berlin,  1895. 


Immunity  acquired  by  natural  means  447 

Yaillard1  and  Remlinger2  upon  a  whole  series  of  infective  diseases 
and  intoxications,  such  as  diphtheria,  cholera  peritonitis,  anthrax, 
experimental  typhoid  septicaemia,  etc.,  showed  conclusively  the 
correctness  of  Ehrlich's  results.  Well-vaccinated  males,  even  when 
hypervaccinated,  never  transmit  their  immunity  to  their  descendants. 
This  acquired  property,  like  so  many  others,  is  not  hereditary  in  the 
strict  sense  of  the  word.  The  females,  on  the  other  hand,  with  rare 
exceptions,  transmit  their  acquired  immunity  to  their  young,  but 
this  transmission  can  in  no  way  be  attributed  to  the  ovum  ;  it  is  here, 
then,  no  longer  a  question  of  hereditary  immunity  properly  so  called. 
According  to  Ehrlich  the  female  furnishes  in  her  blood  plasma  the 
antitoxin  which  passes  into  the  circulation  of  the  foetus.  In  all 
respects  this  is  allied  to  the  so-called  passive  immunity  (or  antitoxic 
immunity  of  von  Bearing).  It  is  due  entirely  to  the  direct  intro- 
duction of  antitoxin,  manufactured  by  the  cells  of  the  maternal 
organism,  into  the  body  of  the  progeny.  The  living  elements  of  the 
foetus  play  no  part  in  it,  and  it  is  for  this  reason  that  the  antitoxins 
and  immunity  in  the  new-born  animal  disappear  so  very  rapidly, — 
within  a  few  weeks  after  birth.  Wernicke  accepts  the  views  of 
Ehrlich  in  their  entirety.  He  found  that  the  immunity  of  female 
guinea-pigs  was  transmitted  to  the  new-born  animal ;  but  this  [469] 
hereditary  transmission  was  exhausted  in  a  single  generation  ;  it  was 
not  found  in  the  second  generation.  Wernicke  was  able  to  demon- 
strate that  the  refractory  condition  in  guinea-pigs,  born  of  mothers 
vaccinated  against  diphtheria,  persisted  for  three  months.  Vaillard 
found  that  it  was  retained  in  certain  cases  for  an  even  longer  period, — 
up  to  the  fifth  month.  On  one  occasion  he  even  observed  the 
transmission  of  the  immunity  to  a  second  generation.  A  female 
guinea-pig,  born  of  a  mother  immunised  against  tetanus,  gave  birth 
to  a  young  one  which,  when  tested  a  month  after  birth  with  a  ten 
times  lethal  dose  of  the  toxin,  contracted  merely  a  slight  tetanus. 

From  this  fact,  as  well  as  from  the  fact  that  the  immunity  of  the 
young  ones  bom  of  vaccinated  mothers  persists  longer  than  does  that 
conferred  by  the  injection  of  antitoxic  serum,  Vaillard  concludes 
that  there  exists  a  kind  of  hereditary  immunity  which  is  "fixed"  by 
the  cells.  He  thinks  that  not  only  the  antitoxins  and  other  anti- 
bodies but  also  certain  living  elements,  especially  the  leucocytes,  are 
able  to  pass  from  the  maternal  blood  into  that  of  the  foetus  and  to 

1  Ann.  de  VInst.  Pasteur,  Paris,  1896,  t.  x,  p.  65. 
9  Ann.  de  VInst.  Pasteur,  Paris,  1899,  t.  xin,  p.  129. 


448  Chapter  XIV 

transmit  to  it  the  properties  acquired  by  the  mother.  At  this  point 
we  may  recall  the  facts  demonstrated  by  von  Behring  and  Ransom 
that  antitoxin  persists  much  longer  in  the  blood  of  an  animal 
when  it  is  introduced  with  the  serum  of  the  same  species.  (We 
have  described  these  observations  in  Chapter  xn.)  Now,  since  in 
hereditary  transmission  the  antitoxin  passes  over  with  the  blood 
plasma  of  the  same  species,  whilst  in  the  experiments  on  antitoxic 
immunity  it  is  generally  injected  with  the  serum  of  a  different  species, 
it  is  easy  to  understand  that  the  former  should  persist  for  a  longer 
period  than  the  latter.  It  is,  therefore,  very  probable  that  this 
immunity  of  the  offspring  from  vaccinated  mothers  is  not  in  any  way 
a  case  of  true  hereditary  immunity,  but  is  due  simply,  as  maintained 
by  Ehrlich,  to  the  passage  of  ready  prepared  antibodies  from  the 
mother  to  the  foetus.  In  the  immunities  against  diphtheria  and 
tetanus  we  have  the  direct  passage  of  antitoxins ;  in  transmitted 
immunity  against  infection  by  the  vibrios  of  Koch  and  Gamaleia, 
so  carefully  studied  by  Vaillard,  we  have,  very  probably,  the  passage 
of  corresponding  fixatives  from  the  mother  to  the  foetus. 

Dzierzgowsky1  in  a  recent  study  on  hereditary  immunity  denies 
[470]  the  passage  of  antibodies  and  toxins  through  the  placenta.  He 
thinks  that  the  foetus  does  not  acquire  its  immunity  through  the 
blood  of  the  mother,  but  at  a  very  much  earlier  period.  The  ovum 
contained  in  the  Graafian  follicle  would,  according  to  this  observer, 
come  in  contact  with  a  fluid  very  rich  in  antitoxin,  whence  it  might 
imbibe  the  necessary  amount  of  this  antibody  to  ensure  the  immunity 
of  the  new-born  animal.  Dzierzgowsky  bases  this  opinion  on  experi- 
ments in  which  antidiphtheria  serum  injected  into  pregnant  goats  and 
dogs  did  not  produce  any  antitoxic  power  in  the  blood  of  the  foetus. 
But  in  the  experiments  on  these  animals  the  injections  consisted 
of  the  serum  of  the  horse— a  different  species.  This  must  modify, 
profoundly,  the  conditions  of  the  passage  of  the  antitoxin  through 
the  placenta. 

Dzierzgowsky  made  a  single  experiment  upon  a  mare,  immunised 
with  diphtheria  toxin,  and  its  foal.  Whilst  the  serum  of  the  former 
was  markedly  antitoxic,  that  of  the  foal  did  not  possess  this  property 
in  the  slightest  degree.  Hence  the  conclusion  that  the  antitoxin  of  the 
mother  had  not  passed  into  the  blood  of  the  foetus.  But  the  blood  of 
the  foal  was  not  withdrawn  until  some  ten  months  after  birth.  Now, 
as  the  so-called  hereditary  immunity  only  lasts  for  a  very  short  time 
1  Arch.  d.  tici.  biul.,  St  Petersbourg,  1901,  t  vm,  p.  211. 


Immunity  acquired  ty  natural  means  449 

Dzierzgowsky's  experiment  supplies  no  evidence  against  the  passage 
of  antitoxin  through  the  placenta. 

In  order  to  prove  that  the  immunity  against  toxins  may  really 
be  acquired  by  the  ovum,  Dzierzgowsky1  carried  out  a  series  of 
experiments  with  the  eggs  of  fowls  immunised  against  diphtheria 
toxin.  The  yolk  of  the  egg,  in  accordance  with  the  discovery  made  by 
F.  Klemperer,  contained  antitoxin ;  and  this  antitoxin  passed  into 
the  blood  of  the  hatched  chickens.  These  facts,  though  in  themselves 
very  interesting,  cannot  be  used  to  refute  the  view  that  antitoxins 
pass  through  the  mammalian  placenta.  It  is  true  that  this  view  is 
perhaps  not  yet  completely  proved,  but  it  accords  well  with  all  the 
known  facts.  Thus,  the  frequent  presence  of  diphtheria  antitoxin 
in  the  blood  of  new-born  infants  is  explained  much  better  on  the 
assumption  that  it  passes  through  the  placenta  than  that  it  is  due 
to  an  immunisation  of  the  ovum  surrounded,  in  the  Graafian  follicle, 
by  antitoxic  fluid.  It  is  difficult  to  conceive  how  this  immunity  could 
be  so  fully  retained  during  the  nine  months  of  pregnancy. 

In  support  of  his  interpretation  of  the  phenomenon  of  immunity  [471] 
transmitted  by  the  mother  to  her  progeny  Ehrlich  invokes  his 
beautiful  discovery  of  the  immunity  conferred  by  the  maternal  milk. 
A  vaccinated  female  is  capable  of  communicating  to  her  young 
a  portion  of  the  antibodies  manufactured  in  her  organism,  not  only 
by  the  blood  channels,  but  also,  in  certain  cases,  by  the  milk  with 
which  she  feeds  her  young. 

The  transmission  of  antitoxins  by  milk  has  been  demonstrated  by 
Ehrlich,  and  this  has  since  been  confirmed  by  many  observers  (see 
Chapter  xn).  When  Ehrlich  found  that  the  immunity  of  the  progeny 
is  retained  for  a  longer  time  than  is  that  which  is  conferred  by  in- 
jections of  antitoxic  serum,  he  conceived  the  idea  of  investigating 
whether  the  cause  of  more  prolonged  retention  did  not  reside  in 
the  transmission  of  the  maternal  antitoxin  by  the  milk.  With  the 
object  of  verifying  this  he  took,  at  the  moment  when  they  had  given 
birth  to  young,  unvaccinated  mice  and  mice  that  had  been  vaccinated 
against  various  toxins  (ricin,  abrin,  tetanotoxin).  He  so  changed  the 
progeny  that  the  vaccinated  mothers  nourished  the  young  born  of  the 
normal  mice,  whilst  the  normal  mothers  suckled  the  offspring  of  the 
vaccinated  mice.  The  result  of  these  ingenious  and  delicate  experi- 
ments fully  confirmed  his  anticipations.  The  vaccinated  mice  trans- 
mitted their  immunity  not  only  to  the  young  ones  to  which  they  had 

1  Arch.  d.  Sci.  bioL,  St  Petersbourg,  1901,  t.  vni,  p.  421. 
B.  29 


450  Chapter  XIV 

given  birth  but  also  to  those  they  had  merely  nourished  with  their  milk. 
This  observation  proved,  to  demonstration,  that  the  antitoxins  are 
absorbed  by  the  alimentary  canal,  a  very  important  fact  from  several 
points  of  view.  Later  researches  have  shown  that  only  very  young 
mice  are  capable  of  assimilating  antitoxin  through  the  intestinal  wall. 
Adult  mice,  fed  by  Ehrlich  with  quantities  of  antitoxic  milk,  acquired 
neither  immunity  nor  any  antitoxic  property  of  the  blood.  Later, 
Vaillard  (L  c.)  was  able  to  show  that  even  the  young  of  other  species 
of  animals  such  as  the  guinea-pig  and  the  rabbit  are  incapable  of 
appropriating  the  antitoxins  from  milk  by  the  alimentary  canal.  He 
repeated  Ehrlich's  experiments  with  new-born  guinea-pigs  and  rabbits 
which  he  caused  to  be  suckled  by  mothers  vaccinated  against  tetanus. 
These  young  rodents,  so  treated,  were  found  to  possess  no  immunity 
whatever ;  they  were  not  able,  therefore,  to  absorb  the  antitoxin 
[472] found  in  the  milk  of  their  nurses.  Remlinger  (I.e.)  made  similar 
experiments  with  young  guinea-pigs  and  rabbits  suckled  by  foster- 
mothers  which  had  been  vaccinated  against  the  coccobacillus  of 
typhoid  fever.  As  in  Vaillard's  experiments,  the  result  was  negative, 
the  milk  of  the  foster  mother  did  not  communicate  any  refractory 
condition  to  the  nurselings.  Remlinger  drew  the  same  conclusion 
from  his  researches  on  the  transmission  of  the  agglutinative  property 
of  the  body  fluids.  When  female  rabbits  and  guinea-pigs  are  vac- 
cinated during  gestation  the  young  ones  acquire,  along  with  the 
immunity  against  the  typhoid  coccobacillus,  a  certain  agglutinative 
power  of  the  blood  serum.  When,  however,  these  vaccinated  females 
suckle  the  progeny  of  non-vaccinated  mothers  the  agglutinative 
power  of  the  milk  of  the  foster  mother  never  passes  into  the  blood  of 
the  nurselings.  Some  years  before  this,  Widal  and  Sicard1  had 
demonstrated  the  same  fact  that  young  rabbits  and  new-born  kittens, 
when  fed  with  agglutinative  milk,  acquired  no  power  of  agglutinating 
the  typhoid  coccobacillus.  They  agreed  with  Ehrlich,  however,  that 
the  blood  serum  of  young  mice  fed  with  agglutinative  milk  acquired 
the  power  of  agglutinating  the  typhoid  micro-organism. 

As  it  was  important  to  determine  whether  the  human  subject  was 
capable  of  acquiring  a  certain  immunity  by  absorbing  antibodies 
contained  in  the  milk,  the  study  of  this  question  was  taken  up, 
especially  from  the  point  of  view  of  agglutinative  power.  Although 
the  relations  of  this  agglutinative  power  with  immunity  are  very 
problematical  it  would  be  interesting,  bearing  in  mind  the  analogy 
1  Compt.  rend.  Soc.  de  biol.,  Paris,  1897,  p.  804. 


Immunity  acquired  fy  natural  means  451 

between  the  agglutinative,  antitoxic,  and  protective  properties,  to 
ascertain  whether  the  ingestion  of  agglutinative  milk  can  confer  any 
agglutinative  property  on  the  blood  serum.  Numerous  researches  in 
this  direction  were  carried  out  in  connection  with  typhoid  fever. 
Widal  and  Sicard  (I.e.)  caused  a  person  to  drink  daily  (for  a  period  of 
three  weeks)  half  a  litre  of  milk  coming  from  an  immunised  goat,  a 
milk  which  powerfully  agglutinated  the  typhoid  coccobacillus.  The 
blood,  examined  on  several  occasions,  never  showed  the  slightest 
agglutinative  power.  This  experiment  goes  to  prove  that,  in  the 
adult  human  subject,  the  agglutinin  does  not  pass  from  the  ali- 
mentary canal  into  the  circulation.  May  it  not  perhaps  be  otherwise 
in  infants  which  are  fed  on  milk  only  ?  An  observation  by  Landouzy 
and  Griffon1  seemed  to  confirm  this  supposition.  They  first  demon- 
strated the  agglutinative  power  of  the  blood  serum  in  a  woman  who 
had  contracted  typhoid  fever  three  months  after  her  lying-in.  Being  [473] 
a  mild  attack  the  woman  continued  to  suckle  her  child  during  the 
whole  course  of  the  fever.  On  examination  of  the  blood  of  the  infant 
it  was  found  that  the  serum  agglutinated  the  micro-organism  of 
typhoid  fever.  These  observers  did  not  measure  the  agglutinative 
power  of  the  blood,  either  in  the  infant  or  in  the  mother.  This 
omission  deprives  their  observation  of  value  since  it  is  now  recognised 
that  normal  human  blood  fairly  frequently  exhibits  some  power  of 
agglutinating  the  typhoid  coccobacillus.  For  diagnostic  purposes  it 
is  necessary,  therefore,  always  to  measure  this  power  in  order  to  be 
sure  that  it  is  higher  than  that  of  the  normal  blood. 

It  is  all  the  more  difficult  to  draw  any  positive  conclusion  from 
the  observations  of  Landouzy  and  Griffon  because  in  several  similar 
cases  the  result  has  been  entirely  different.  Thus  Achard  and 
Bensaude2  have  shown  that  the  blood  of  an  infant,  suckled  by  a 
nurse  attacked  by  typhoid  fever  and  whose  serum  became  distinctly 
agglutinative,  was  incapable  of  bringing  about  clumping  of  the 
typhoid  coccobacilli.  Schumacher3,  working  in  FraenkeFs  laboratory 
in  Halle,  studied  a  case  with  very  great  care.  A  woman  gave  birth 
at  full  term  to  an  infant  whose  blood  serum  exhibited  a  certain 
agglutinative  power.  The  mother  suckled  the  infant  from  its  birth. 
Although  her  milk  manifested  a  very  considerable  agglutinative 
property,  the  blood  of  the  child  exhibited  not  only  no  increase  in 

1  Compt.  rend.  Soc.  de  biol.,  Paris,  1897,  p.  950. 

2  Semaine  med.,  Paris,  1896,  p.  303. 

3  Ztschr.f.  Hyg.,  Leipzig,  1901,  Bd.  xxxvir,  S.  323. 

29-2 


452  Chapter  XIV 

agglutinative  power  but  a  marked  diminution.  The  agglutinin  of  the 
maternal  blood  had  not  passed  into  the  fluids  of  the  child. 

From  the  point  of  view  of  the  impossibility  of  acquiring  immunity 
by  suckling,  therefore,  the  human  subject  may  be  grouped  with  the 
guinea-pig,  rabbit  and  cat.  Up  to  the  present  the  mouse  is  the  only 
exception.  It  would  be  very  important,  with  the  object  of  finding  a 
means  of  communicating  immunity  by  way  of  the  intestine,  to  study 
the  precise  conditions  which  govern  this  phenomenon.  In  hereditary 
immunity,  or  rather  in  what  appears  to  be  such,  those  cases  where 
the  new-born  animal  exhibits  a  resisting  power  induced  by  the 
vaccination  to  which  it  has  been  subjected  in  the  womb  of  the  mother 
must  be  borne  in  mind.  We  have  already  cited  the  example  given 
by  Remlinger  of  rabbits  and  guinea-pigs  born  refractory  against  the 
typhoid  coccobacillus,  which  had  been  injected  into  the  mother 
[474]  animals.  In  those  cases  where  the  vaccination  of  the  mothers  has 
been  carried  out  during  the  period  of  gestation  the  immunity  of  the 
young  ones  is  more  permanent  than  when  it  was  completed  before 
that  period.  Into  this  same  group  come  those  cases  where  women, 
vaccinated  during  the  course  of  pregnancy,  give  birth  to  infants 
refractory  to  vaccine.  Similar  facts  have  been  reported  by  veterinary 
surgeons  with  regard  to  sheep-pox  ;  Arloing,  Cornevin,  and  Thomas1 
have  offered  similar  demonstrations  with  regard  to  symptomatic 
anthrax. 

These  results  may  be  more  or  less  closely  associated  with  those 
where  the  child  attacked  by  an  infective  disease  immunises  the 
mother.  Such  facts  are  rare.  We  know  that  a  healthy  mother  may 
give  birth  to  a  syphilitic  child ;  the  affected  father  introducing  the 
virus  with  the  sperm,  the  contaminated  foetus  contracts  the  disease 
and  the  new-born  infant  is  syphilitic.  According  to  Ehrlich  and 
Hubener  (L  c.  p.  54),  the  foetus  instead  of  infecting  the  mother  sets 
up  in  her  a  refractory  condition.  It  must  be  confessed  that  as  yet  we 
do  not  understand  the  mechanism  of  this  immunity ;  but  in  any  case 
we  have  here  to  do  with  an  example  of  immunity  naturally  acquired 
under  very  special  conditions. 

Here  again  must  be  recognised  another  form  of 'immunisation  : — 
where  the  child  born  of  a  syphilitic  mother  remains  healthy  and 
contracts  syphilis  neither  by  the  breast  nor  through  the  kisses  of  the 
mother.  Here,  undoubtedly,  we  have  an  immunity  against  syphilis 
acquired  in  the  womb  of  the  mother,  who  may,  however,  readily  com- 
1  "Le  charbon  bacterien,"  Paris,  ISbo,  p.  184. 


Immunity  acquired  by  natural  means  453 

municate  her  disease  to  other  persons  by  means  which  are  without 
effect  on  her  own  infant.  This  example  comes  under  the  law  of 
Profetta.  Here  again  the  mechanism  of  the  acquired  immunity  is 
absolutely  unknown. 

It  must  be  admitted  that,  generally,  we  are  still  very  imperfectly 
informed  concerning  immunity  as  acquired  by  natural  paths.  In 
cases  where  this  immunity  is  developed  as  the  result  of  an  attack  of 
an  infective  disease  the  phenomena  observed  closely  resemble  those 
that  have  been  observed  after  vaccination  by  living,  fully  active,  or 
attenuated  viruses,  by  micro-organisms  which  have  been  killed,  or  by 
the  products  of  these  micro-organisms.  These  vaccinations  which 
bring  about  isopathic  (von  Behring)  or  active  (Ehrlich)  immunity  give 
rise  to  transient  and  mild  diseases  and  are  confined  almost  completely 
to  the  diseases  contracted  by  natural  means  which  terminate  in  [475] 
recovery  and  give  rise  to  a  refractory  condition.  The  immunisation 
of  the  foetus  comes  into  the  same  series. 

On  the  other  hand,  the  immunity  which  was  believed  to  be 
hereditary  and  which  results  merely  from  the  direct  passage  of  the 
antibodies  of  the  blood  or  of  the  milk  of  the  mother  to  the  foetus 
and  to  the  child  come  into  a  group  of  cases  characterised  by  what 
Ehrlich  has  termed  a  condition  of  passive  immunity.  We  have 
already  discussed  (Chapter  x)  the  thesis  that  this  term  "  passive  "  is 
applicable  only  in  rare  cases.  Most  frequently  it  is  necessary  that 
the  living  cells  of  the  organism  which  receives  the  antibodies — 
antitoxin,  fixatives  or  others— should  contribute  their  quota  in  order 
to  ensure  the  refractory  condition.  This  rule  is  undoubtedly  applic- 
able to  the  examples  of  immunity  acquired  by  the  new-born  progeny 
of  unaffected  mothers. 


[476]  CHAPTER  XV 


PROTECTIVE  VACCINATIONS 

Vaccinations  against  I.  Small-pox. — II.  Sheep-pox.— III.  Rabies.— IV.  Rinderpest. — 
V.  Anthrax.— VI.  Symptomatic  Anthrax.— VII.  Swine  Erysipelas. — VIII.  Pleuro- 
pneumonia  in  the  Bovidae.— IX.  Typhoid  Fever.— X.  Plague.— XI.  Tetanus. — 
XII.  Diphtheria. 

IN  the  preceding  chapters  I  have  attempted  to  present  to  the 
reader  a  general  view  of  the  phenomena  of  immunity  against  infec- 
tive micro-organisms  and  against  their  toxic  products.  I  shall  now 
attempt  to  give  a  review  of  the  facts  acquired  in  connection  with 
the  prevention  of  the  infective  diseases  of  man  and  of  the  chief 
domestic  animals  by  means  of  vaccination.  Vaccinations  as  we 
know  can  be  carried  out  either  with  viruses  the  constituents  of 
which  have  not  as  yet  been  recognised,  with  micro-organisms  grown 
on  various  nutrient  media,  with  virulent  or  attenuated  micro-organ- 
isms, or  with  microbial  products  deprived  of  the  micro-organisms  by 
which  they  have  been  built  up.  In  addition  to  these  methods  we 
may  vaccinate  with  protective  or  antitoxic  serum  and  other  body 
fluids,  with  normal  serum,  or  with  a  whole  series  of  fluids  not 
excepting  water. 

I.  Vaccination  against  small-pox. — We  naturally  commence  the 
series  with  vaccination  against  small-pox,  which  is  one  of  the  oldest 
and  one  of  the  best  known,  having  been  practised  in  every  country 
in  Europe  for  more  than  100  years.  Small-pox,  a  very  contagious 
and  fatal  malady,  was  very  rife  in  the  18th  century.  Large  cities  like 
London  and  Paris  were  severely  affected.  One-tenth  of  the  total 
mortality  was  due  to  this  disease.  According  to  statistical  informa- 
tion, very  exact  for  that  epoch,  the  deaths  from  small-pox  in  London 
[477]  during  the  course  of  the  second  half  of  the  century  (1751 — 1800) 
numbered  more  than  100,000  (102,112)  persons.  During  the  first 


Protective  vaccinations  455 

half  of  the  same  century  this  disease  caused  great  ravages  in  France, 
especially  in  Paris,  where,  according  to  certain  statistics  (Haeser), 
about  14,000  persons  died  in  1716. 

Variolisation  or  "inoculation"  coming  to  Europe  from  the  East, 
had  come  into  extensive  use  when,  at  the  end  of  the  18th  century, 
the  discovery  was  made  that  cow-pox,  the  varioliform  disease  of  the 
Bovidae,  produced  in  persons  who  milked  cows  suffering  from  this 
eruption  an  immunity  against  small-pox.  This  idea,  popular  in 
origin,  was  known  to  breeders  in  England,  France,  Germany,  and 
Holland ;  we  have  thus  an  indication  that  this  knowledge  must  date 
from  a  fairly  distant  period.  Jenner  gave  the  question  a  scientific 
and  experimental  basis,  and  it  was  only  after  his  intervention  that 
vaccination  by  the  contents  of  the  pustules  of  cow-pox  began  to 
spread  more  generally.  During  the  19th  century  an  immense  amount 
of  material  bearing  on  this  question  was  collected ;  we  have  thus 
been  enabled  to  attain  absolutely  exact  results,  and  that  in  spite  of 
the  very  imperfect  state  of  our  knowledge  on  the  etiology  of  small- 
pox and  of  cow-pox.  Long  ago  Chauveau1  demonstrated  that  the 
virus  of  these  diseases  must  be  organised,  because  that  of  the  vaccine 
would  not  pass  through  a  filter.  This  organism  has  been  carefully 
sought,  but  sought  in  vain  in  spite  of  all  improvements  in  micro- 
biological methods.  It  was  thought  that  the  cocci  so  often  found  in 
the  contents  of  the  vaccinal  pustule  was  the  specific  micro-organism 
of  cow-pox.  Such  was  the  opinion  of  the  illustrious  botanist  Cohn2. 
It  was  soon  shown,  however,  that  this  was  not  the  case.  The  cocci, 
principally  staphylococci,  are  "  secondary"  micro-organisms  which  may 
be  absent  from  the  vaccine  without  its  losing  anything  of  its  action. 
A  search  was  then  made  for  the  micro-organism  of  the  vaccine 
among  the  protozoan  organisms.  L.  Pfeiffer3  announced  the  dis- 
covery of  a  species  of  vaccinal  Amoeba.  Guarnieri4  has  even 
described  various  stages  in  the  reproduction  of  this  hypothetical 
parasite  ;  but  Salmon5  demonstrated,  in  a  work  carried  out  in  the  [478] 
Pasteur  Institute,  that  we  had  here  to  deal  merely  with  leucocytes 
which  had  entered  epithelial  cells  and  had  there  undergone  marked 

1  Compt.  rend.  Acad.  d.  sc.,  Paris,  1868,  t  LXVI,  pp.  289,  317,  359. 
a   Virchovfs  ArcMv,  1872,  Bd.  LV,  S.  229. 

3  Monatssch.f.prakt.Dermat.j  Hamburg,  1887;  "Die  Protozoen  als  Krankheits- 
erreger,"  Jena,  1891,  S.  184. 

4  Arch,  per  le  sc.  med.,  Torino,  1892,  t.  xvi,  p.  403. 

5  Ann.de  FInst.  Pasteur,  Paris,  1897,  t.  xi,  p.  289. 


456  Chapter  XV 

degeneration.  Funek1  thought  that  he  was  able  to  confirm  the 
discovery  of  the  sporozoon  of  vaccinia,  but  his  error  was  easily 
demonstrated  (Podwyssozki  and  Mankowski)2.  Up  to  the  present, 
then,  we  have  no  knowledge  of  either  the  micro-organism  of  small- 
pox or  of  that  of  vaccinia.  We  still  employ,  as  formerly,  the  virus 
taken  from  the  vaccinal  pustule.  Even  the  relations  which  exist 
between  the  two  viruses  and  the  two  diseases  which  they  have  set  up 
have  not  yet  been  settled.  Several  authors  believe  that  the  bovine 
disease  is  only  a  modified  and  attenuated  form  of  human  small-pox  ; 
whilst  others  maintain  that  we  have  two  very  different  exanthemata, 
one  of  which — cow-pox — is  capable  of  setting  up  immunity  not  only 
against  itself  but  also  against  small-pox. 

For  a  long  time,  in  order  to  vaccinate  against  small-pox,  the  con- 
tents of  the  vaccinal  pustules  which  formed  on  the  human  subject 
after  an  original  inoculation  of  the  virus  of  cow-pox  were  employed. 
But  a  number  of  cases  of  infection  by  syphilitic  virus  and  certain 
other  accidents  caused  this  method  to  be  abandoned.  A  number  of 
years  ago,  however,  there  spread  throughout  Europe  and  into  several 
countries  of  other  continents  another  method,  which  consists  in 
vaccination  by  "animal  lymph/'  that  is  to -say,  by  the  contents  of 
pustules  developed  on  the  skin  of  the  calf.  This  method  was  first 
earned  out  at  Brussels  in  1868,  under  the  direction  of  Warlomont,  at 
the  Institute  founded  by  the  Belgian  Government  for  the  preparation 
of  vaccine.  The  original  virus  came  from  a  genuine  case  of  cow-pox 
and  has  since  been  kept  up  by  uninterrupted  passage  from  calf  to 
calf.  The  virus  is  introduced  into  the  shaved  skin  of  the  region 
between  the  groin  and  the  udder  as  far  forward  as  the  umbilicus.  It 
is  inoculated  superficially  into  the  epidermis  by  cuts  one  centimetre 
long.  At  the  points  of  inoculation  characteristic  pustules  develop ; 
from  these  the  vaccinal  content  is  withdrawn,  on  the  fifth  day  in 
summer  or  the  sixth  in  winter.  The  contents  are  removed  by 
pressure  and  by  scraping  the  pustules.  The  scrapings  are  mixed 
with  water  and  glycerine.  The  vaccine  thus  prepared  is  put  into 
small  glass  tubes  which  are  sealed  at  both  ends.  This  method,  with 
slight  modifications,  has  extended  to  many  other  countries,  and  is 
[479]  carried  out  either  in  private  establishments  or  in  State  institutions 
as  in  Germany.  For  the  purpose  of  purifying  the  vaccine  it  is 

1  Deutsche  med,  Wchnschr.,  Leipzig,  1901,  S.  130;  Brit.  Med.  Journ.,  London 
1901,  Vol.  i,  p.  44S. 

2  Deutsche  med.  Wchnschr.,  Leipzig,  1901,  S.  261. 


Protective  vaccinations  457 

diluted  and  then  allowed  to  sediment  or  it  may  be  subjected  to 
centrifugalisation.  The  object  of  these  measures  is  to  rid  the 
"lymph"  of  the  micro-organisms  which  accompany  it.  This  object 
is,  however,  only  imperfectly  attained  and  is  moreover  accompanied 
by  an  attenuation  of  the  vaccinal  action.  On  the  other  hand,  pre- 
cautions are  taken  to  ensure  all  possible  cleanliness  during  the 
operation  of  inoculation  and  whilst  the  calves  are  under  treatment. 
Thus,  great  care  is  taken  to  disinfect  the  area  of  inoculation  with 
alcohol  or  some  other  antiseptic  and  to  dress  the  pustules  during  the 
course  of  their  development.  Similarly  the  arms  of  the  patient  to 
be  vaccinated  are  well  washed  ;  following  in  this  the  rules  of  asepsis 
rather  than  of  antisepsis  for  fear  that  the  vaccinal  virus  might  be 
destroyed  by  antiseptic  substances.  Various  instruments  are  made  use 
of  for  vaccination  and  care  is  taken  to  sterilise  these  before  they  are 
used.  Sometimes  the  lancet  is  used,  sometimes  "  plumes  k  vaccin  " 
or  vaccinostyles,  or  a  bistoury  of  iridio-platinum  (Lindenborn)  etc. 

When  the  vaccine  is  of  good  quality  and  the  operation  of 
vaccination  is  well  done,  there  is  no  doubt  as  to  the  protective 
result  obtained  against  small-pox.  The  observations  that  have 
been  collected  for  a  great  number  of  years  past,  in  many  countries, 
place  this  beyond  doubt.  There  are,  indeed,  statistics  from  which 
it  is  impossible  to  draw  any  precise  conclusions  because  they  are 
founded  upon  too  scanty  figures  or  deal  with  conditions  that  are 
too  complex.  This  is  the  case  with  the  Swiss  vaccinations.  Certain 
cantons  (such  as  Zug  and  Uri)  have  made  vaccination  obligatory, 
whilst  others  (Bern,  Zurich,  Lucerne,  etc.)  some  years  ago  abolished 
the  law  which  compels  the  vaccination  of  all  children  in  infancy. 
It  happened  that  for  some  years  small-pox  had  more  victims  in  the 
cantons  of  the  first  group  than  in  those  of  the  second.  The  opponents 
of  antivariolic  vaccination  attempted  to  use  this  as  an  argument 
against  the  utility  of  this  method.  But  a  more  detailed  study  of 
the  facts  clearly  shows  that  it  is  impossible  to  draw  from  it  any 
conclusion  whatever.  Even  in  those  cantons  where  vaccination  is 
supposed  to  be  compulsory  this  law  is  not  carried  out  rigorously, 
and  the  number  of  persons  vaccinated  often  does  not  exceed  that  in 
the  cantons  where  it  is  not  obligatory. 

In  order  to  gain  some  idea  of  the  utility  of  vaccinations  we 
must  collect  statistics  on  a  much  larger  scale  than  are  those  obtain- 
able from  the  Swiss  cantons.  Germany  furnishes  such  statistics. 
Compulsory  vaccination  was  introduced  there  more  than  a  quarter 


458  Chapter  XV 

[480]  of  a  century  ago  (1874),  and  statistical  information  has  been 
collected  with  great  care.  With  the  exception  of  a  slight  increase 
during  the  period  from  1879  to  1885  small-pox  has  diminished 
progressively  since  the  proclamation  of  the  new  law,  and  has  become 
so  rare  that  in  1897  there  were  only  5  fatal  cases  in  the  whole  German 
Empire.  In  the  space  of  13  years  (1886—1898),  in  a  population 
which  embraces  two-fifths  of  the  total  inhabitants  of  the  German 
Empire,  there  were  altogether  five  fatal  cases  of  small-pox  occurring 
in  persons  who  had  been  successfully  revaccinated.  Moreover,  the 
majority  of  the  cases  of  small-pox  occurred  in  the  maritime  towns 
or  in  the  vicinity  of  the  frontier  of  the  Russian  Empire. 

Specially  favourable  results  have  been  obtained  in  the  German 
army,  in  which,  even  before  the  law  of  1874,  vaccination  was  com- 
pulsory. In  25  years  there  occurred  in  the  Prussian  army  only  two 
cases  of  death  from  small-pox.  In  summing  up  the  statistical  data 
on  vaccination  Kubler1,  from  whom  we  have  borrowed  the  above 
statements,  expresses  himself  as  follows  :  "  The  history  of  small-pox 
must  in  all  cases  register  the  fact  that  this  dreaded  disease  has,  as 
the  result  of  general  vaccination,  not  only  become  rare  in  the  German 
Empire  but  that  it  has  almost  completely  disappeared"  (p.  365). 
The  example  of  Germany  encouraged  several  other  countries  to 
introduce  compulsory  vaccination,  and  Roumania,  Hungary,  and 
Italy  have  in  turn  promulgated  similar  laws.  Here  also  it  was  not 
long  before  satisfactory  results  were  obtained.  In  Italy  especially  the 
mortality  from  small-pox  has  largely  decreased  in  recent  years. 

In  England,  where  compulsory  vaccination  was  introduced  some 
time  ago,  it  was  abolished  in  1898.  As  the  opposition  of  the 
people  became  more  manifest,  the  law,  although  it  continued  to 
exist  formally,  was  carried  out  very  imperfectly.  The  number  of 
unvaccinated  children  had  gradually  increased  in  such  a  fashion  that 
in  London  itself  in  1897 — 1898  it  attained  the  proportion  of  24'9  %, 
whilst  in  certain  provincial  districts  it  has  oscillated  between  78'4 
and  86'4°/o-  Under  these  conditions,  the  abolition  of  the  law  of 
compulsory  vaccination  was  only  the  legal  sanction  of  an  accom- 
plished fact.  According  to  the  details  which  have  been  supplied  to 
me  by  the  Jenner  Institute  in  London  (which  has  taken  in  hand  the 

[481]  distribution  of  vaccine),  vaccinations  since  they  are  no  longer  com- 
pulsory have  become  more  frequent  in  England,  and  the  quantity 

1  "  Die  Geschichte  der  Pocken  und  der  Impfung,"  von  Coler's  Bibliothek,  Berlin, 
1901. 


Protective  vaccinations  459 

of  vaccine  distributed  has  increased  considerably.  This  quantity, 
however,  is  not  adequate  because  small-pox  has  again  made  its 
appearance  in  London  in  the  form  of  a  pretty  serious  epidemic1. 

In  France  a  law  is  being  framed  which  will  render  infant  vac- 
cination compulsory.  Up  to  the  present  this  has  not  been  the 
case,  and  small-pox  from  time  to  time  causes  considerable  ravages, 
as  we  may  see  at  this  moment  in  Paris.  During  recent  years  the 
mortality  from  small-pox  in  France  has  been  from  90  to  100  times 
greater  than  in  Germany.  It  is  greater  amongst  the  female  popula- 
tion than  amongst  males  ;  this  constitutes  a  fresh  argument  in  favour 
of  vaccination.  Although  not  compulsory  for  the  whole  of  the 
French  population,  it  is  so  for  soldiers  and  for  children  who  carry  on 
their  studies  in  schools,  and  it  is  for  this  reason  that  small-pox  is 
rarer  amongst  males.  The  most  complete  demonstration  of  this  is 
found  in  the  incidence  of  small-pox  in  the  French  army.  In  spite 
of  a  less  numerous  contingent  of  troops  (451,941 — 457,677)  the 
mortality  from  small-pox  was  greater  during  the  period  when 
vaccination  was  not  yet  carried  out  generally  (1885—1887)  than 
during  the  period  (1889 — 1896)  when  it  was  rigorously  enforced 
on  a  much  larger  number  of  soldiers  (524,733 — 564,643).  From 
13*6  fatal  cases  per  year  in  the  first  period  the  annual  figure 
fell  to  6. 

It  follows,  when  we  take  into  consideration  the  whole  of  the  very 
numerous  data  at  our  disposal,  that  the  usefulness  of  vaccination 
followed  by  revaccination  after  some  (5 — 7)  years  cannot  be  seriously 
called  in  question.  As  to  the  inconveniences  that  may  be  caused, 
they  are  observed  in  very  rare  cases,  and  then  most  frequently  when 
impure  vaccines  are  used,  or  when  the  vaccinated  skin  becomes 
contaminated.  According  to  the  German  statistics  there  were 
registered  in  the  space  of  13  years  (1885 — 1897),  in  32  millions 
of  vaccinations,  113  fatal  cases  as  the  result  of  infection  of  the 
wounds.  In  forty-six  of  these  it  was  proved  that  the  small  wound 
had  been  contaminated  by  impurities  introduced  by  those  attending 
on  them.  The  remaining  67  fatal  cases  could  be  ascribed  to  the 
vaccines  themselves.  We  must,  however,  still  regard  these  cases  as  too 
numerous  and  as  being  readily  avoidable  by  the  adoption  of  rigorous 
asepsis.  To  sum  up,  the  anti-variolous  vaccination  by  the  virus  of 
cow-pox  constitutes  a  method  of  very  great  value  in  the  prevention  of  [482] 
one  of  the  most  dreaded  of  infective  diseases,  but  it  is  evident  that 
1  Lancet,  London,  1901,  Vol.  n,  p.  796. 


460  Chapter  XV 

improvement  can  still  be  made  in  this  branch  of  practice.  If  science 
should  succeed  some  day,  as  we  may  be  permitted  to  hope  it  will,  in 
finding  the  micro-organism  of  vaccinia  and  of  small-pox,  and  it  should 
succeed  in  growing  it  in  pure  media,  it  might  react  very  beneficially 
on  the  practical  application  of  vaccination.  The  more  simple  the 
methods,  the  less  chance  will  there  be  of  the  occurrence  of  those 
unsuccessful  cases  which,  even  now,  are  rare  exceptions. 

IT.  Vaccinations  against  sheep-pox  (la  claveUe}.— Sheep-pox, 
being  a  disease  very  similar  to  human  small-pox  and  very  serious 
from  an  economic  point  of  view,  the  idea  was  conceived  of  fighting  it 
by  methods  similar  to  those  used  against  small-pox.  Since  the  18th 
century  there  has  been  practised  on  a  large  scale  the  artificial  im- 
munisation of  sheep  by  the  inoculation  of  the  virus  of  the  sheep-pox 
(clavelisation)  just  as  the  variolisation  of  man  was  practised  before 
the  discovery  of  cow-pox.  For  this  purpose  it  was  necessary  to  have 
a  considerable  quantity  of  virus ;  this  was  obtained  by  inoculating 
sheep-pox  into  the  skin  of  sheep.  This  inoculation  was  effected  either 
with  a  lancet  or,  according  to  Soulie's  method1,  by  means  of  a  Pravaz 
syringe.  The  pustules,  developed  under  these  conditions,  were  gene- 
rally of  large  size  and  capable  of  furnishing  a  considerable  quantity 
of  the  virulent  lymph  (claveau)  used  for  immunisation.  This  fluid, 
when  gathered  pure,  and  kept  in  a  closed  vessel  protected  from  light 
and  heat,  retains  its  virulence  for  a  long  time :  unlike  what  is  observed 
in  the  case  of  vaccine,  the  addition  of  glycerine  destroys  the  virulence 
of  the  lymph  pretty  quickly.  For  use,  the  lymph  is  diluted  with  ten 
times  its  volume  of  2%  borated  water;  the  fluid  thus  obtained 
is  inoculated  into  the  extremity  of  the  tail  or  of  the  ear ;  usually 
a  pustule,  which  remains  single,  is  formed  at  the  point  of  inoculation. 
Clavelisation  rarely  sets  up  a  generalised  eruption  which  is  always 
serious  and  sometimes  fatal. 

In  France  the  law  ordains  the  clavelisation  of  flocks  in  which 

sheep-pox  appears;    but  it  interdicts  its   practice   in   unattacked 

flocks ; — it  is  easy  to  understand  the  reason  for  this ;  in  infected 

flocks  all,  or  almost  all,  the  sheep,  gradually  become  ill  and  the 

[483]  illness  lasts  for  some  time  ;  clavelisation  diminishes  both  the  duration 

and  the  gravity  of  the  disease  ;  the  mortality  that  it  causes,  although 

sometimes  very  great,  the  French  sheep  being  very  susceptible  to 

sheep-pox,  is  always  much  less  than  that  due  to  a  natural  contagion ; — 

1  Medecine  moderne,  Paris,  1896,  p,  441 


Protective  vaccinations  461 

on  the  other  hand,  the  clavelisation  of  a  healthy  flock,  beyond  the 
fact  that  it  may  cause  considerable  losses,  is  attended  by  the  special 
danger  that  it  creates  centres  from  which  the  contagion  may  invade 
all  the  flocks  of  the  district 

But  there  are  countries  in  which  protective  and  general  claveli- 
sation does  not  present  these  inconveniences — the  countries  where 
the  disease  is  endemic  and  where  the  sheep  are  very  resistant  to  the 
action  of  its  virus.  This  is  the  case  in  Algeria ;  sheep-pox  exists 
there  permanently  without  doing  much  damage ;  but  the  Algerian 
sheep,  which  take  sheep-pox  without  suffering  any  apparent  illness, 
communicate  to  French  sheep  amongst  which  they  are  introduced 
a  very  malignant  sheep-pox  which  sometimes  kills  as  many  as  50  per 
cent,  of  the  flock.  This  explains  and  justifies  the  measures  recently 
taken  by  the  Minister  of  Agriculture,  forbidding  the  importation  of 
Algerian  sheep  into  France  unless  they  have  been  vaccinated  at  least 
a  month  previously. 

In  many  other  countries  clavelisation  is  likewise  enacted,  being 
authorised  in  cases  where  it  may  be  very  useful  and  interdicted 
in  other  cases.  In  certain  countries,  e.g.  Germany,  Holland,  and  Den- 
mark, clavelisation  can  be  put  into  force  by  the  Government,  which 
alone  has  the  right  to  authorise  it  under  certain  circumstances.1 

III.  Antirabic  vaccinations.  Vaccination  against  rabies  has  this 
point  in  common  with  those  against  small-pox  and  sheep-pox,  that 
it  is  effected  with  a  virus  whose  micro-organism  is  as  yet  unknown. 
On  the  other  hand,  it  is  distinguished  by  its  efficacy  during  the 
incubation  period.  When  persons  are  vaccinated  during  the  incu- 
bation period  of  small-pox,  or  sheep  during  the  same  period  of 
sheep-pox,  the  vaccinations  by  vaccine  and  claveau  are  incapable  of 
arresting  the  disease  and  the  infections  continue  to  follow  their 
normal  course.  "When,  on  the  other  hand,  we  vaccinate  men  or 
animals  that  have  been  bitten  by  mad  animals  or  inoculated  with  the 
rabic  virus  by  other  means,  the  antirabic  vaccination,  with  rare 
exceptions,  prevents  the  development  of  rabies.  This  vaccination, 
taking  advantage  of  the  length  of  the  incubation  period  of  rabies, 
constitutes,  therefore,  a  special  type,  intermediate  between  pro-  [484] 
tective  vaccination,  properly  so  called,  and  a  therapeutic  method 
of  treatment. 

1  Nocard  et  Leclainche,  "Les  maladies  microbiennes  des  animaux,"  2e  edition, 
Paris,  1898,  pp.  464,  469. 


462  Chapter  XV 

It  is  to  Pasteur  that  science  and  humanity  owe  the  invention 
of  this  method.  Aided  by  his  collaborators,  especially  by  Roux,  he 
established  in  the  first  place  a  whole  series  of  important  facts  on  the 
subject  of  the  rabic  virus  and  of  experimental  rabies.  He  then  set 
himself  to  elaborate  a  practical  method  capable  of  preventing  the 
manifestation  of  the  disease  in  dogs  inoculated  with  rabic  virus  and 
in  men  bitten  by  mad  animals.  He  succeeded  in  solving  this  problem 
in  1885. 

Pasteur's  antirabic  vaccines  are  prepared  from  the  spinal  cords 
of  rabbits  that  have  died  of  experimental  rabies  as  the  result  of 
the  inoculation  of  the  virus  bearing  the  name  of  "fixed  virus." 
Prepared  in  the  laboratory,  this  virus  presents  the  characteristic 
feature  that  when  inoculated  under  the  dura  mater  of  rabbits  it  sets 
up  in  them  the  first  rabic  manifestations  after  an  incubation  period 
of  six  or  seven  days.  The  disease  soon  assumes  the  typical  paralytic 
form  which  lasts  several  days.  Whilst  the  period  of  incubation 
presents  only  very  limited  variation,  the  time  of  death  is  subject  to 
much  greater  variation,  especially  according  to  the  season  of  the  year. 
Sometimes  the  rabbits  will  die  on  the  eighth  day  after  the  inoculation 
of  the  virus :  but  death  may  be  delayed  one  or  two  days,  rarely 
more. 

It  is  necessary  to  wait  for  the  natural  death  of  the  mad  rabbits 
before  the  spinal  cord  is  extracted,  and  not  to  kill  them  before  this 
term,  for  it  is  only  during  the  final  moments  of  life  that  the  rabic 
virus  is  abundant  and  is  distributed  uniformly  through  the  whole 
substance  of  the  organ.  After  removal  from  the  vertebral  canal  the 
cord  is  suspended  in  glass  vessels  containing  solid  potassium  hydrate 
at  the  bottom.  A  whole  series  of  cords  so  prepared  are  then  kept  in  a 
dark  chamber  heated  to  23°  C.  or  thereabouts.  The  progressive  desic- 
cation which  the  cords  undergo  under  these  conditions  diminishes  their 
virulence.  At  the  end  of  several  days  of  this  treatment  the  desiccated 
cord,  instead  of  producing  rabies  in  6 — 7  days  in  rabbits  inoculated 
under  the  dura  mater  by  trepanning,  induces  it  after  longer  periods 
of  incubation.  Finally,  the  cords  do  not  produce  even  the  slightest 
symptoms  of  the  disease. 

The  fundamental  basis  of  the  Pasteurian  method  consists  in  the 

fact  that  the  desiccated  cord,  inoculated  as  an  emulsion  below  the 

skin  of  animals,    produces    in    them    a   complete    and   permanent 

[485]  immunity   against  inoculation  of  the    most    powerful    rabic  virus 

beneath  the  dura  mater.    This  experiment,  frequently  repeated  on 


Protective  vaccinations  463 

rabbits  and  dogs,  justified  Pasteur  in  1885  in  attempting  the  first 
vaccinations  of  persons  bitten  by  rabid  animals,  especially  dogs.  The 
encouraging  results  of  these  early  attempts  led  to  the  foundation 
of  the  Pasteur  Institute  in  Paris,  devoted,  in  part,  to  antirabic  vac- 
cinations. Shortly  afterwards,  antirabic  Institutes  were  founded  in 
many  other  European  towns,  and  later  in  North  and  South  America, 
in  Indo-China,  the  East  Indies,  and  in  Africa.  At  present  there  are 
in  France  six  such  Institutes  (Paris,  Lille,  Marseilles,  Montpellier, 
Lyons,  Bordeaux),  in  Russia  9,  in  Italy  6,  etc.  The  last  of  these 
institutions  founded  in  Europe  is  that  of  Berlin,  where  it  forms  a 
branch  of  the  Institute  for  Infective  Diseases  carried  on  under  the 
direction  of  Robert  Koch.  The  foundation  of  an  antirabic  institute 
in  Berlin  had  a  very  important  significance  from  several  points  of 
view.  In  the  first  place,  it  indicates  the  definite  acceptance  of  the 
Pasteurian  method,  a  method  which  has  been  discussed  so  long  and 
so  keenly.  Secondly,  it  proves  that  even  in  a  State  where  there  is  a 
highly  organised  sanitary  police,  antirabic  vaccinations  may  still  be 
of  great  service. 

Seeing  that  it  was  in  the  Pasteur  Institute  of  Paris  that  the 
method  of  antirabic  vaccinations  was  first  elaborated  and  that  it 
has  undergone  a  very  prolonged  ordeal,  the  method  there  used 
serves  as  a  model  for  the  practice  of  almost  all  other  institutes. 
Although  in  some  of  them  methods  which  differ  more  or  less  from 
the  original  may  have  been  introduced,  the  fundamental  principle 
upon  which  they  are  based  remains  the  same. 

According  to  the  Pasteurian  method  properly  so  called  the 
vaccinal  inoculations  are  commenced  with  cords  that  have  been 
dried  for  14  days  and  have  thus  lost  their  virulence.  A  piece  five 
millimetres  long  is  pounded  up  with  very  weak  veal  broth.  Up  to 
3  c.c.  of  the  emulsion  thus  prepared  is  injected  below  the  skin  of  the 
flank.  The  same  day  a  second  injection  of  the  same  quantity  of  an 
emulsion  of  a  cord  which  has  been  drying  for  13  days  is  made  at  the 
corresponding  position  on  the  opposite  side.  Each  day  an  advance 
is  made  by  injecting  emulsions  of  cord  which  are  increasingly  fresh 
and  the  treatment  is  concluded  by  the  introduction  of  virulent  cords, 
which  have  been  kept  at  23°  C.  for  3  days  only.  The  ordinary 
medium  treatment  lasts  for  15  clays.  On  the  first  5  days  two  vaccine 
injections  a  day  are  made.  On  the  last  10  days,  when  gradually 
fresher  and  more  virulent  cords  are  employed,  only  a  single  in- [486] 
jection  is  made  each  day.  The  injections  are  made  with  syringes  of 


464  Chapter  XV 

the  Pravaz  type  and  are  carried  out  under  conditions  of  rigorous 
cleanliness. 

If  the  bites  are  numerous,  or  if  they  are  situated  on  exposed  parts, 
the  treatment  is  prolonged  for  18  days  and  is  further  distinguished  in 
that  the  cords  of  4  and  of  3  days  are  injected  much  more  frequently. 

In  especially  grave  cases,  when  the  bites  are  on  the  face  and  head, 
the  treatment  extends  over  3  weeks.  A  more  rapid  progress  is  made 
by  making  four  injections  instead  of  two  during  the  two  first  days ; 
in  this  way  a  greater  quantity  of  the  virulent  cords  is  injected  than 
in  the  first  two  types  of  treatment. 

The  effect  of  the  antirabic  vaccinations  is  usually  very  good. 
During  the  early  years  of  their  application  the  results  were  fully 
discussed  from  all  points  of  view,  and  no  efforts  were  neglected  of 
seeking  out  objections  of  every  kind.  For  the  purpose  of  obtain- 
ing rigorously  accurate  statistics  a  separate  division  was  made,  at 
the  Pasteur  Institute,  for  the  cases  of  persons  treated  after  bites 
inflicted  by  dogs  whose  rabic  condition  had  been  demonstrated 
experimentally  (by  the  injection  of  an  emulsion  of  the  bulb  below 
the  dura  mater  or  into  the  anterior  chamber  of  the  eye  of  the  rabbit 
or  guinea-pig).  A  second  and  special  set  of  statistics  was  drawn  up 
of  cases  where  the  bites  had  been  inflicted  by  animals  whose  rabic 
condition  had  been  recognised  by  veterinary  examination.  Indi- 
viduals bitten  by  animals  that  were  simply  suspected  to  suffer  from 
rabies  were  kept  separate. 

Thanks  to  this  systematic  classification  we  were  able,  at  the  Pasteur 
Institute  of  Paris,  to  establish  the  fact  that  the  antirabic  vaccinations 
performed  on  persons  bitten  by  animals  that  were  undoubtedly  mad 
resulted  in  an  extremely  low  mortality  from  rabies.  Finding  it 
impossible  to  attack  these  results,  demonstrated  with  the  precision  of 
a  laboratory  experiment,  the  adversaries  of  the  Pasteurian  method 
alleged  that,  quite  apart  from  any  vaccination,  the  percentage  of 
cases  of  rabies  in  persons  bitten  by  mad  animals  is  not  greater 
than  amongst  the  vaccinated.  A  hitch  in  the  application  of  the 
new  vaccinal  method  soon  demonstrated  how  entirely  unfounded 
was  this  objection.  At  the  Bacteriological  Institute  of  Odessa, 
founded  in  1886,  that  is  to  say  almost  immediately  after  the  Paris 
Institute,  the  first  attempts  at  vaccination  were  followed  by  a  mor- 
tality from  rabies  of  6*88  per  cent.,  a  figure  incomparably  higher 
than  that  of  the  Paris  Institute.  Analysing  the  probable  causes  of 
[487]  this  want  of  success  it  was  found  that  the  Russian  rabbits,  being 


Protective  vaccinations  465 

much  smaller  than  the  French  ones,  furnished  far  too  small  an  amount 
of  vaccinal  matter.  This  being  the  case,  the  introduction  of  a  more 
intensive  treatment  was  sufficient  to  cause  the  mortality  to  drop 
suddenly  to  0*8  per  cent.  This  fact,  added  to  so  many  other  proofs, 
finally  convinced  the  most  sceptical  and  brought  about  a  general 
acceptance  of  the  Pasteurian  method. 

In  course  of  time  the  number  of  cases  observed  has  become  very 
considerable  and  the  experience  gained  in  the  manipulation  of  this 
method  very  wide.  The  improvements  made  in  the  details  of  the 
vaccinal  practice  have  brought  about  a  progressive  diminution  in 
the  mortality  amongst  the  persons  treated.  From  0*94  per  cent,  in 
1886  the  mortality  (counted  from  the  16th  day  after  the  completion 
of  the  vaccinations)  fell  in  1897  to  0*39  per  cent.,  in  1900  to  0'28  per 
cent.  In  the  space  of  15  years  (1886—1900)  there  have  been  treated 
in  Paris  24,665  persons,  of  whom  107  died  from  rabies,  giving  an 
average  of  0*43  per  cent.1.  The  greatest  mortality  was  registered 
during  the  early  years  of  the  application  of  the  method,  and  the 
rate  of  the  later  years  (1896—1900)  oscillated  between  0'39  per  cent, 
and  0'20  per  cent. 

The  results  obtained  in  the  majority  of  the  other  antirabic  insti- 
tutes corroborate  those  of  the  Pasteur  Institute  of  Paris.  Thus, 
according  to  the  latest  statistics  of  the  St  Petersburg  Institute2,  the 
mortality,  in  1899,  among  persons  who  had  completed  their  vac- 
cinations, was  about  0'5  per  cent.  At  Berlin3  there  were  treated 
during  the  same  period  384  persons,  of  whom  2  died  from  rabies 
during  treatment,  whilst  a  third  succumbed  on  the  14th  day  after 
the  close  of  the  vaccinations.  Only  this  latter  case  ought,  ac- 
cording to  the  principles  generally  accepted,  to  be  counted  as  an 
unsuccessful  case,  this  would  give  a  mortality  of  0*26  per  cent. 

Quite  recently,  the  antirabic  treatment  has  been  so  reinforced  that 
the  treatment  terminates  with  the  injection  of  cords  desiccated  for 
two  days  or  even  one  day  only.  The  results  of  this  intensive  treat- 
ment have  not  yet  been  reported  upon. 

According  to  the  statistics  of  the  Berlin  Institute  rabies  is  far 

1  Report  by  Viala  in  the  Ann.  de  I'Inst.  Pasteur,  Paris,  1901,  t.  xv,  p.  445. 
There  will  be  found  in  Marie's  work,  "La  rage"  (Collection  des  aides-mem.,  Paris, 
1900),  many  details  on  antirabic  vaccination. 

2  According  to  Krajonchkine,  in  the  Arch.  d.  Sci.  biol.,  St  Petersbourg,  1901, 
t.  viu,  p.  349. 

3  According  to  Marx  in  Klin.  Ja/irb.,  Berlin,  1900,  Bd.  vn,  S.  1. 

B.  30 


466  Chapter  XV 

[488]  from  being  so  rare  in  Germany  as  was,  at  one  time,  generally  sup- 
posed. During  the  year  1899  its  presence  was  demonstrated,  by  the 
experimental  method,  in  206  dogs  coming  from  various  districts.  It 
is  in  Silesia,  Western  Prussia,  and  Posen  that  rabies  in  dogs  has  been 
observed  most  frequently. 

Antirabic  vaccinations  have  also  been  performed  on  herbivorous 
animals  (sheep,  goats,  cattle,  and  horses)  which  are  immunised  by 
means  of  injections  of  the  rabic  virus  into  the  veins,  according  to  the 
method  suggested  by  Nocard  and  Roux1,  as  the  result  of  experi- 
ments made  by  Galtier2. 

IV.  Vaccinations  against  rinderpest.  For  some  time  attempts 
were  made  to  find  a  means  of  immunising  the  Bovidae  and  other 
ruminants,  susceptible  to  rinderpest,  against  this  terrible  disease, 
which  causes  great  ravages  in  regions  where  it  is  endemic  and  greater 
still  in  those  regions  where  it  only  appears  in  epidemic  form.  The 
good  results  obtained  from  "  clavelisation "  suggested  the  idea  of 
immunising  against  rinderpest  by  the  inoculation  of  the  rinderpest 
virus,  but  all  such  attempts  gave  unsatisfactory  results,  the  inocula- 
tion setting  up  a  rinderpest  as  grave,  and  often  as  fatal  as  the  natural 
disease.  Only  in  recent  years  have  we  succeeded  in  elaborating 
methods  of  vaccination  really  capable  of  coping  effectively  with 
rinderpest.  Koch3  went  to  Cape  Colony,  where  this  disease  had 
recently  appeared  and  had  caused  enormous  losses,  with  the  intention 
of  finding  a  practical  method  of  arresting  the  scourge.  In  spite 
of  his  technique  and  incomparable  skill  he  was  as  unsuccessful  in 
finding  the  parasite  of  rinderpest  as  had  been  other  investigators. 
The  micro-organism  of  this  disease  remains  unknown.  It  was 
necessary,  however,  to  seek  a  remedy  against  it.  Koch,  studying 
the  properties  of  the  bile  of  animals  that  had  died  from  rinderpest, 
recognised  that  the  injection  of  this  bile  into  normal  animals  con- 
ferred upon  them  a  fairly  certain  immunity,  and  this  fact  served  as 
the  basis  on  which  to  work  out  a  practical  method  of  combating 
rinderpest  on  a  large  scale.  At  first  this  method  was  received  with 
[489]  much  enthusiasm,  but  experience  soon  demonstrated  the  incon- 
veniences it  often  presented.  Kolle  and  Turner4,  who  continued  the 

1  Ann.  de  VInst.  Pasteur,  Paris,  1888,  t.  n,  p.  341. 

2  Compt.  rend.  Acad.  d.  sc.,  Paris,  1881,  t.  xcin,  p.  284. 

3  Deutsche  med.  Wchnschr.,  Leipzig,  1897,  SS.  225,  241. 

4  Ztschr.f.  Hyg.,  Leipzig,  1898,  Bd.  xxix,  S.  309. 


Protective  vaccinations  467 

researches  on  rinderpest  in  Cape  Colony,  extolled  Koch's  method  at 
the  commencement  of  the  epidemic  with  the  object  of  establishing 
around  the  original  disease  centre  an  unaffected  zone  which  would 
interfere  with  the  propagation  of  the  disease.  They  recognised, 
however,  that  this  method  could  not  be  employed  generally,  for  the 
reason  that  it  does  not  set  up  immunity  until  the  end  of  eight  days, 
during  which  period  the  animals  may  contract  the  disease.  Further, 
it  demands  the  sacrifice  of  a  large  number  of  animals  in  order  to 
provide  the  vaccinal  bile  required  for  the  vaccinations  ;  finally,  it 
confers  an  immunity  of  short  duration  only  (four  to  six  months). 

It  was  necessary,  therefore,  to  find  some  method  that  was  more 
generally  applicable.  With  this  object  Koch  himself  began  to  study 
the  blood  serum  of  animals  that  had  recovered  spontaneously  from 
rinderpest.  He  was  able  to  assure  not  only  himself,  but  several  other 
observers,  that  this  serum  was  capable  of  rendering  normal  animals 
into  which  it  is  injected  refractory.  Bordet  and  Danysz,  who  studied 
rinderpest  in  the  Transvaal  in  1897,  made  many  experiments  in  this 
direction  and  devised  a  method  which  gave  good  results  in  practice. 
But  it  was  left  to  Kolle  and  Turner  to  work  out  a  method  at  once 
simple  and  easily  applied,  one  which  soon  came  into  general  use. 
This  method  is  known  by  the  name  of  "  simultaneous  vaccinations/' 
It  consists  in  the  injection  of  a  protective  serum  simultaneously  with 
the  virulent  blood.  To  prepare  the  former  the  authors  just  men- 
tioned made  use  of  animals  that  had  recovered  spontaneously  from 
rinderpest  or  of  Bovidae  that  had  been  immunised  by  bile  or  by  some 
other  method.  It  was  recognised  that  the  protective  power  of  the 
serum  of  animals  that  have  recovered  is  very  small  and  cannot  confer 
immunity  on  normal  animals,  except  when  injected  in  large  doses. 
Kolle  and  Turner  showed  that  if  Bovidae  that  have  recovered  spon- 
taneously are  injected  with  very  large  quantities  of  virulent  blood 
coming  from  animals  fatally  attacked,  the  protective  power  of  the 
serum  of  the  former  is  markedly  increased  and  a  serum  is  obtained 
which  is  active  in  small  doses  and  which  gives  good  results  in  prac- 
tice. This  serum  may  be  kept  for  a  long  time  by  the  addition  of  a 
small  quantity  of  carbolic  acid.  The  immunity  conferred  by  this 
serum  upon  normal  animals  is  immediate,  but  of  short  duration;  it  [490] 
is  completed  by  making  a  simultaneous  injection  of  virulent  blood ; 
we  thus  obtain  a  double  immunity,  one  part  immediate,  the  other  per- 
manent ;  to  get  this  result,  however,  the  serum  must  not  be  mixed 
with  the  virulent  blood,  for  when  this  is  done  the  immunity  conferred 

30—2 


468  Chapter  XV 

is  trifling  or  nil.  On  the  other  hand,  it  is  complete  and  persists  for 
several  months  when  the  protective  serum  is  injected  separately  on 
one  side  of  the  body  and  the  virulent  blood  on  the  other. 

Kolle  and  Turner  had  to  defend  their  method  against  many  ill- 
founded  objections  and  attacks,  but  they  succeeded  in  getting  it 
accepted,  not  only  in  Cape  Colony  but  also  in  many  other  parts  of 
Africa,  and  in  many  countries  in  Europe  and  in  Asia.  In  1898  it  was 
decided  at  a  conference  which  met  in  Cape  Town  to  use  the  method  of 
simultaneous  vaccinations  to  the  exclusion  of  all  others.  This  method 
has  since  been  applied  on  a  very  large  scale  and  it  was  not  long  before 
favourable  results  were  obtained.  The  same  method  has  proved  to 
be  very  successful  with  Nicolle  and  Adil-Bey1  of  Constantinople, 
who  now  prepare  large  quantities  of  the  antirinderpest  serum,  and 
combat  this  disease  with  great  success  in  the  Ottoman  empire. 
Yersin2  adopted  the  same  method  to  fight  the  cattle  plague  in  Indo- 
China,  where  it  causes  great  ravages,  especially  among  buffaloes.  His 
Institute  at  Nha-Trang  has  become  a  centre  for  the  preparation  of 
the  specific  serum,  which  he  distributes  over  a  vast  territory.  In  the 
East  Indies  the  simultaneous  method  has  been  applied  by  Rogers3. 
In  Russia,  where  rinderpest  is  endemic  in  many  regions,  the  Institute 
of  Experimental  Medicine  at  St  Petersburg  furnishes  the  serum 
destined  to  prevent  the  propagation  of  this  epizootic  disease4. 

In  a  few  years  this  method  of  simultaneous  vaccination  has  been 
extended  to  all  the  countries  ravaged  by  rinderpest  and  has  already 
rendered  immense  services  to  agriculture. 

V.  Anti-anthrax  vaccinations.  In  the  first  four  sections  of  this 
Chapter  we  have  brought  together  the  methods  which  have  as  their 
[491]  basis  the  vaccination  by  viruses  whose  nature  is  as  yet  unknown. 
Since  we  cannot  obtain  them  by  artificial  culture,  we  have  to  intro- 
duce them  with  animal  fluids  : — either  the  contents  of  vaccinal  or 
clavelar  pustules,  or  matter  from  rabic  nervous  centres,  or  again  the 
blood  of  animals  attacked  by  rinderpest.  In  the  case  last  mentioned, 
in  order  to  prevent  the  too  serious  effect  of  the  injection  of  the  virus, 
it  is  combined  with  a  simultaneous  injection  of  protective  serum. 

1  Ann.  de  VInst.  Pasteur,  Paris,  1899,  t.  xm,  p.  319  ;  1901,  t  xv,  p.  715. 

2  Rec.  de  med.  vet.,  Paris,  1901,  pp.  48,  115. 

3  Report  on  an  experim.  Investig.  of  the  method  of  Inoculation  against  Rinder- 
pest, Calcutta,  1900 ;  Ztschr.f.  Hyg.,  Leipzig,  1900,  Bd.  xxxv,  S.  59. 

4  Nencki,  Sieber  and  Wyznikiewicz,  Arch,  internat.  de  Pharmacodyn.,  Gand  et 
Paris,  1899,  vol.  v,  p.  475. 


Protective  vaccinations  469 

In  the  case  of  the  vaccinations  against  anthrax  we  pass  to  the 
group  of  viruses  whose  organised  nature  is  well  known  and  which 
can  be  injected  in  pure  culture  grown  on  artificially  prepared  media. 
This  method  constitutes  one  of  Pasteur's  most  brilliant  discoveries, 
made  in  collaboration  with  Chamberland  and  Roux.  Before  they  had 
found  a  satisfactory  method  of  vaccinating  against  anthrax  these 
observers  had  to  solve  the  problem  in  connection  with  a  less  com- 
plicated and  less  difficult  case.  From  the  first,  in  his  studies  on 
pathogenic  micro-organisms,  Pasteur  had  devoted  his  attention  to 
finding  a  means  of  communicating  immunity  against  these  parasites. 
With  the  aid  of  Chamberland  and  Roux  he  was  not  long  in  discover- 
ing a  method  by  which  it  was  possible  to  attenuate  the  virulence  of 
the  micro-organism  of  fowl  cholera  and  to  vaccinate  fowls  against 
this  terrible  disease  by  inoculating  them  with  this  attenuated 
micro-organism.  Guided  by  these  results  Pasteur,  Chamberland  and 
Roux  set  to  work  to  find  the  vaccine  against  anthrax ;  they  were  soon 
confronted  by  a  serious  obstacle  in  the  formation  of  spores  which 
prevented  the  attenuation  of  the  bacilli.  This  obstacle  they  overcame 
by  submitting  cultures  of  the  bacillus  to  a  temperature  of  42*5°  C. 
Under  this  condition  spores  do  not  develop,  and  the  bacilli  become 
attenuated  at  the  end  of  a  longer  or  shorter  period.  Although  in 
possession  of  these  attenuated  viruses,  it  still  needed  very  laborious 
investigations  to  adapt  them  to  the  vaccination  of  various  species  of 
animals  susceptible  to  anthrax,  especially  sheep.  In  this  they  were 
also  successful,  and  in  1881,  over  20  years  ago,  Pasteur  and  his  collabo- 
rators demonstrated  the  efficacy  of  their  method  on  a  large  number 
of  animals.  This  demonstration  was  made  at  Pouilly-le-Fort  before 
a  large  commission.  We  may  affirm  that  this  celebrated  experiment 
opened  a  new  path  to  science  and  to  the  practice  of  vaccination. 
It  was  performed  on  50  sheep,  half  of  which  were  vaccinated  twice 
with  twelve  days'  interval,  the  other  25  sheep  serving  as  control 
animals.  Fourteen  days  after  the  vaccination  by  the  second  vaccine  [492] 
all  the  50  sheep  were  subjected  to  a  test  inoculation  of  a  very  strong 
anthrax  virus.  Two  days  later  the  vaccinated  animals  remained 
unaffected,  whilst  the  control  animals  had  all  succumbed  to  anthrax. 

Similar  experiments,  undertaken  in  France,  Hungary,  Germany, 
Russia  and  elsewhere,  confirmed  the  efficacy  of  anthrax  vaccinations 
and  led  to  their  extension  into  all  the  countries  where  bacterial 
anthrax  was  rife.  From  the  year  1881  the  method  came  into  regular 
use,  and  before  the  end  of  that  year  there  had  been  vaccinated,  in 


470  Chapter  XV 

France  alone,  62,000  sheep  and  6,000  Bovidae.  Since  these  first 
attempts,  made  on  a  large  scale,  gave  such  good  results,  the  anti- 
anthrax  practice  was  not  long  in  spreading  through  France,  then 
into  Hungary  and  several  other  European  countries.  Later,  it 
extended  into  other  continents,  especially  into  South  America 
(Argentina)1  and  Australia.  Vaccinations  against  anthrax  were  also 
applied  to  horses  with  the  same  good  results2. 

In  France  the  anti-anthrax  vaccines  are  prepared  at  and  sent 
out  from  the  Pasteur  Institute  of  Paris.  These  vaccines  consist  of 
broth  cultures  of  attenuated  bacilli,  of  which  the  weakest,  the  first 
vaccine,  is  fatal  to  the  mouse  and  small  guinea-pigs.  The  bacilli  of 
the  second  vaccine  are  less  attenuated,  and  are  capable  of  killing 
not  only  adult  guinea-pigs  but  even  a  certain  number  of  rabbits, 
when  inoculated  subcutaneously.  The  two  vaccines  are  races  of  the 
anthrax  bacillus,  capable  of  producing  spores  which  present  the  same 
degree  of  virulence  as  the  filamentous  bacilli  which  gave  them  birth. 

The  anti-anthrax  vaccines  are  sent  out  in  tubes  containing  the 
quantity  necessary  for  the  vaccination  of  a  large  number  of  animals. 
The  vaccinations  are  made  especially  in  spring  in  order  that  the 
animals  may  be  protected  during  the  hot  season,  which  is  usually 
more  favourable  to  the  development  of  anthrax  epidemics. 

In  the  sheep  the  vaccines  are  injected  below  the  skin  on  the 
inner  aspect  of  the  thigh.  One-eighth  of  a  c.c.  of  the  first  vaccine 
is  injected  with  a  somewhat  modified  Pravaz  syringe.  Twelve  or 
[493]  fifteen  days  later  a  similar  injection  is  made  on  the  opposite  side  with 
the  second  vaccine.  In  the  Bovidae  the  vaccines  are  injected  behind 
the  shoulders,  where  the  skin  is  thinnest.  In  the  horse  the  injections 
must  be  made  on  the  sides  of  the  neck  and  shoulders.  In  large 
mammals  double  the  amount  (^th  of  a  c.c.)  of  each  vaccine  is 
injected. 

The  tubes  of  vaccine,  once  opened,  should  not  be  employed  a 
second  time.  Care  must  be  taken  to  use  the  whole  of  their  contents 
at  one  series  of  vaccinations. 

The  vaccinal  injections  produce  tumefaction  at  the  point  of 
inoculation  and  are  followed  by  a  slight  rise  of  temperature.  But 
these  symptoms  are  of  little  importance  and  soon  disappear.  Serious 
complications  and  fatal  results  from  the  vaccinations  are  very  rare. 

1  J.  Mendez,  Anal.  d.  Circ.  Med.  Argent.,  Buenos  Aires,  1901,  t.  xxiv,  Nos.  5,  6. 

2  On  the  methods  of  vaccination  against  anthrax  see  Chamberland,  *'  Le  charbon 
et  la  vaccination  charbonneuse,"  Paris,  1883. 


Protective  vaccinations  471 

The  loss  due  to  these  accidents  is  estimated  at  one-half  per  cent, 
in  sheep  and  a  quarter  per  cent,  in  the  Bovidae. 

The  refractory  condition  resulting  from  the  vaccination  requires 
for  its  development  a  period  of  about  a  fortnight.  The  immunity  is 
then  very  substantial  and  lasts  for  a  fairly  long  time.  According  to 
Chamber-land  60°/o  of  the  sheep  retain  their  immunity  a  year  after 
they  have  been  vaccinated.  But  as  a  great  number  of  animals 
then  become  susceptible,  it  is  usual  to  revaccinate  annually. 

According  to  the  statistics  furnished  by  the  vaccine  department 
of  the  Pasteur  Institute  there  have  been  vaccinated,  up  to  the  1st 
of  January  1900,  a  total  of  4,971,494  sheep,  and  708,980  cattle. 
Abroad  the  corresponding  figures  are  3,831,948  and  1,869,445.  Alto- 
gether, the  number  of  animals  vaccinated  amounted  to  11,381,867, 
of  which  3,626,206  have  been  treated  with  the  vaccine  furnished  by 
the  Budapest  Laboratory. 

The  results  of  the  anti-anthrax  vaccinations  were  found  to  be  so 
favourable  that  it  was  unnecessary  to  introduce  any  improvements 
in  technique.  Attempts  have  certainly  been  made  to  prepare  anti- 
anthrax  serums,  and  these  have  been  successful,  but  up  to  the  present 
such  serums  have  not  been  introduced  into  practice. 

VI.  Vaccinations  against  symptomatic  anthrax.  Symptomatic 
anthrax,  which  is  often  confounded  with  true  anthrax,  is  set  up,  as 
demonstrated  by  Arloing,  Coruevin,  and  Thomas,  by  a  specific  anae- 
robic micro-organism  to  which  has  been  given  the  name  of  Bacillus 
chaavaeL  Immediately  after  the  discovery  of  the  attenuation  of  [494] 
viruses  and  of  vaccines  against  fowl  cholera,  the  three  observers 
above  mentioned  tried  to  apply  it  to  symptomatic  anthrax.  Finally 
they  devised  a  method  which  was  soon  adopted  in  practice,  and 
which,  for  nearly  twenty  years,  has  been  used  in  the  vaccination  of 
the  Bovidae  in  countries  where  symptomatic  anthrax  is  most  preva- 
lent. This  is  especially  the  case  in  mountainous  districts,  such  as 
Switzerland,  the  Bavarian  Alps,  the  Dauphine,  L'Auvergne,  etc. 

Arloing,  Cornevin,  and  Thomas1  prepare  two  vaccines  against 
symptomatic  anthrax  by  a  method  very  different  from  that  used  in 
the  preparation  of  the  Pasteurian  anti-anthrax  vaccines.  They  take 
the  virus  from  the  muscles  invaded  by  the  micro-organism ;  they 
trit iimte  a  piece  of  the  tumefied  muscle  in  a  mortar,  adding  to  it  a  few 

1  "Le  charbon  baeterien,"  Paris,  1883  ;  2e  editicm,  1887. 


472  Chapter  XV 

drops  of  water.  The  mixture  is  filtered  through  muslin  and  the  fluid 
dried  at  37°  C. ;  a  virulent  brown  powder  is  thus  obtained.  In  the 
preparation  of  the  vaccines  a  portion  of  this  powder  is  mixed  with 
water  and  subjected  to  a  temperature  of  100° — 104°  C.  for  seven 
hours.  Another  portion  is  heated  during  the  same  number  of  hours 
to  90° — 94°  C.  only.  This  latter  forms  the  second  vaccine  whilst  the 
first  portion  constitutes  the  first. 

In  practice  the  two  vacciual  powders  are  dissolved  in  cooled  boiled 
water  and  are  introduced  into  the  subcutaneous  tissue  of  the  animals 
that  it  is  wished  to  immunise.  The  second  vaccine  should  be  injected 
8  to  12  days  after  the  first.  The  vaccines  are  usually  tolerated  very 
well  by  the  Bovidae  and  confer  upon  them  a  definite  and  permanent 
immunity.  In  spite  of  certain  drawbacks  this  method,  known  as  the 
"Lyons  method,"  has  proved  to  be  a  very  serviceable  one  and  is 
retained  as  the  best  devised  up  to  the  present.  Its  efficacy  is 
proved  by  the  fact  that  in  the  period  from  1884  to  1895  in  400,000 
vaccinated  animals  the  mortality  has  only  been  1  per  1 ,000.  Arloing, 
Cornevin,  and  Thomas  thought  that  raising  the  virus  to  a  high 
temperature  brought  about  a  real  attenuation. 

Leclainche  and  Vallee1,  who  have  recently  returned  to  the  study 
of  this  question,  have  shown  that  this  view  cannot  be  maintained. 
[495]  In  reality  the  spores,  after  being  heated  to  90° — 1 04°  C.,  gave  rise  to 
bacilli  endowed  with  their  normal  and  complete  virulence.  But  the 
heating  in  the  preparation  of  the  Lyons  vaccines  destroys  the  toxin 
manufactured  by  the  Bacillus  chauvaei,  with  the  result,  that  the 
spores  now  become  the  prey  of  phagocytes  :  it  is  for  this  reason  and 
for  this  reason  alone  that  the  inoculation  of  these  vaccines  is  so  well 
tolerated.  All  the  spores  of  the  vaccinal  powder  are  not  eaten  by  the 
phagocytes :  those  which  are  found  in  the  centre  of  solid  particles 
of  the  powder  offer  a  prolonged  resistance  to  the  action  of  the  cells, 
and  some  of  them  germinating  produce  bacilli  and  give  rise  to  a  mild 
disease  capable  of  conferring  immunity.  The  germination  of  these 
spores  is  further  facilitated  by  the  presence  of  foreign  micro- 
organisms in  the  vaccinal  powders  ;  these  organisms  help  to  interfere 
with  the  phagocytosis  of  the  spores  of  symptomatic  anthrax. 

In  the  course  of  their  researches,  Leclaiuche  and  Vallee  demon- 
strated that  it  is  easy  to  vaccinate  animals  susceptible  to  anthrax 
and  to  confer  on  them  a  substantial  immunity  by  means  of  a  single 
protective  injection  of  a  pure  culture  of  Bacillus  chauvaei.  For  this 
1  Ann.  de  I'lnst.  Pasteur,  Paris,  1900,  t.  xiv,  pp.  202,  513. 


Protective  vaccinations  473 

purpose  they  use  cultures  grown  in  broth  made  from  the  pig's  stomach 
("  bouillon  de  panse  "  or  Martin's  broth)  which  they  heat  for  2  hours 
at  70°  C.  The  cultures,  so  treated  and  injected  in  quantities  of 
1  to  2c.c.  into  Bovidae,  induce  in  them  an  immediate  immunity. 
These  authors  are  persuaded  that  the  vaccination  by  this  method 
might  be  used  on  a  large  scale  with  certain  advantages  over  the 
method  at  present  in  use.  A  single  injection,  instead  of  two,  involves 
a  great  economy,  and  the  injection  of  pure  vaccinal  cultures  obviates 
the  accidents  caused  by  the  foreign  organisms  which  are  found  mixed 
with  the  Lyons  vaccine. 

On  the  other  hand,  Leclainche  and  Valle*e  think  that  vaccination 
by  serums  has  no  future  in  the  fight  against  symptomatic  anthrax 
and  should  only  be  used  in  exceptional  cases. 

It  is  evident  that  the  Lyons  method  is  capable  of  being  improved 
and  some  day  may  be  replaced  by  another.  Still  it  must  be  remem- 
bered that  it  has  already  preserved  a  very  great  number  of  animals 
from  certain  death  by  symptomatic  anthrax. 

VII.  Vaccinations  against  swine  erysipelas.  Swine  erysipelas  is 
a  disease  widely  distributed  in  nearly  all  countries  where  the  breeding 
of  pigs  is  carried  on  on  a  large  scale.  It  is  a  very  fatal  disease,  and  it  is 
estimated  that  in  France  alone  at  least  100,000  pigs  of  the  value  of 
more  than  five  million  francs  succumb  to  it  annually.  Unfortunately  [496] 
swine  erysipelas  is  often  confounded  by  breeders  with  other  epizootic 
diseases,  especially  pneumo-enteritis  of  the  pig.  This  confusion  has 
often  resulted  in  large  losses  to  agriculture. 

Soon  after  the  vaccinations  against  anthrax  became  a  part  of 
veterinary  practice,  Pasteur1,  assisted  by  Thuillier,  took  up  the  study 
of  swine  erysipelas  which  was  causing  great  ravages  in  the  department 
of  Vaucluse.  They  were  not  long  in  discovering  that  the  true  cause 
of  the  disease  was  a  very  small  bacillus  capable  of  growing  in  pure 
culture  in  nutrient  broth.  Guided  by  his  former  investigations, 
Pasteur  with  his  collaborator  undertook  minute  researches  into  the 
reinforcement  and  attenuation  of  the  virulence  of  the  bacillus  of  swine 
erysipelas  which  led  them  to  the  elaboration  of  a  method  of  vaccina- 
tion capable  of  conferring  on  pigs  a  high  degree  of  protection  against 
the  disease.  Following  the  line  of  the  anthrax  vaccinations,  Pasteur 
and  Thuillier  prepared  two  vaccines  against  the  erysipelas,  the  first 
more  attenuated  than  the  second.  The  bacilli  of  these  two  vaccines 
1  Compt.  rend.  Acad.  d.  sc.,  Paris,  1883,  t.  xcvn,  p.  1163. 


474  Chapter  XV 

were  cultivated  in  broth  and  sent  out  in  tubes  similar  to  those  em- 
ployed in  the  distribution  of  the  anthrax  vaccines. 

The  vaccines  are  in  themselves  innocuous  and  are  capable  of 
communicating  to  the  inoculated  pig  an  immunity  sufficiently  durable 
to  be  of  real  service.  Young  pigs  being  less  susceptible  to  the 
erysipelas  than  are  the  adults,  it  is  generally  preferred  to  vaccinate 
young  pigs  of  from  two  to  four  months.  The  vaccination  is  done  at 
two  separate  times.  The  first  vaccine,  in  a  dose  of  one-eighth  of  a  cubic 
centimetre,  is  inoculated  subcutaneously  on  the  inner  aspect  of  the 
right  thigh  ;  the  second  vaccine  is  inoculated  in  the  same  way,  12  or 
15  days  later,  into  the  left  thigh.  The  immunity  that  follows  these 
vaccinations  is  not  fully  established  until  the  end  of  the  second  week. 

In  spite  of  the  many  advantages  of  the  Pasteurian  method  the 
vaccinations  against  swine  erysipelas  have  not  spread  so  much  as 
one  might  have  expected ;  and  they  have  found  a  general  applica- 
tion abroad  rather  than  in  France.  It  is  only  necessary  to  cast  a 
glance  at  the  statistics  to  be  convinced  of  this.  From  the  date  of 
the  introduction  of  the  Pasteurian  vaccinations  in  1884  up  to  the 
1st  January,  1900,  there  had  been  vaccinated  in  France  in  all  428,746 
pigs,  whilst  abroad,  where  the  vaccinations  were  introduced  some 
[497]  years  later,  the  number  of  pigs  vaccinated  was  4,819,387-  Of  this 
number  the  great  majority  (4,194,191)  had  been  treated  in  Hungary. 
The  losses  amongst  the  vaccinated  animals  were  insignificant  (1'68  %) 
when  compared  with  an  average  mortality  of  20°/o  amongst  un- 
vaccinated  pigs. 

This  limited  extension  of  the  vaccination  of  pigs  in  France  arises 
from  various  causes.  In  many  countries  the  breeding  is  on  too  small 
a  scale  to  allow  of  the  intervention  of  the  veterinarian  and  of  the 
expenses  which  the  vaccinations  involve.  On  the  other  hand,  it 
cannot  be  denied  that  the  Pasteurian  method  presents  certain  draw- 
backs in  practice.  The  living,  although  attenuated,  bacilli  introduced 
may  sometimes  serve  as  centres  of  infection,  especially  in  cases,  rare 
no  doubt,  where  the  vaccinated  animal  contracts  a  chronic  form  of  the 
disease.  The  Pasteurian  vaccines  must,  therefore,  be  avoided  in  dis- 
tricts where  the  erysipelas  has  not  yet  appeared.  Their  application 
in  countries  already  infected  presents  the  further  drawback  that  the 
immunity  requires  for  its  establishment  a  fairly  long  time,  sufficiently 
long  to  permit  the  micro-organism  to  kill  a  large  number  of  pigs 
before  the  vaccines  have  conferred  any  immunity  upon  them. 

It  is  natural  that,  under  such  conditions,  an  attempt  has  been 


Protective  vaccinations  475 

made  to  replace  the  Pasteurian  method  by  some  other  method  less 
risky.  Hence,  since  the  discovery  of  the  principle  of  sero-therapy 
several  investigators  have  sought  to  apply  it  to  swine  erysipelas. 
Emmerich  and  Mastbaum1  were  the  first  to  demonstrate  that  the 
blood  of  rabbits,  immunised  with  the  bacilli  of  this  disease,  acquire 
a  very  marked  protective  power.  They  have  even  attempted  to  con- 
struct from  the  results  of  their  researches  methods  which  might  be 
applied  practically.  It  is  especially  however  to  Lorenz2,  a  Darmstadt 
veterinarian,  that  we  owe  the  first  practical  application  of  this  method. 
He  prepared  protective  serums  by  injecting  erysipelas  bacilli  into 
rabbits  and  pigs,  and  demonstrated  that  the  inoculation  of  these 
serums,  when  combined  with  that  of  the  living  bacilli,  conferred  upon 
pigs  a  sufficient  immunity  and  one  that  was  set  up  immediately  after 
the  introduction  of  the  serum.  According  to  Lorenz's  method  it  is  first 
necessary  to  give  a  protective  injection  of  serum  ;  some  days  (3 — 5) 
afterwards  this  is  followed  by  an  inoculation  of  living  bacilli  coming 
from  the  attenuated  erysipelas  known  in  Germany  under  the  name  of  [498] 
"  Backsteinblattern."  About  two  weeks  later  a  further  injection  of 
the  same  bacilli,  but  in  double  quantity,  is  given.  This  method,  there- 
fore, involves  three  vaccinal  injections  as  against  two  in  the  Pasteurian 
method.  It  is  consequently  dearer  than  the  latter,  but,  as  it  presents 
certain  undeniable  advantages,  an  attempt  was  made  to  introduce 
it  into  veterinary  practice.  But  being  much  more  complicated  en- 
deavours were  made  to  simplify  it.  Yoges  and  Schiitz,  by  methods 
which  have  remained  secret,  soon  obtained  a  more  active  serum, 
and  finally  Leclainche3  of  Toulouse,  after  demonstrating  that  the 
horse  is  the  best  animal  for  the  production  of  a  very  active  serum, 
succeeded  in  devising  a  method  of  vaccination  as  simple  as  it  was 
effective.  He  gave  to  it  the  name  of  "  serum-vaccinations."  The  first 
inoculation  is  made  with  a  mixture  of  specific  serum  and  a  culture  of 
living  and  virulent  bacilli.  This  inoculation  is  well  borne  by  all  pigs 
and  may  be  made  without  any  regard  to  the  age  of  the  animal  The 
immunity  is  set  up  immediately  after  the  injection  of  the  mixture,  but 
it  is  not  sufficiently  durable  for  the  requirements  of  practice.  For 
this  reason  Leclainche  followed  up  the  first  injection  by  a  second,  which 

1  Arch.f.  Hyg.,  Miinchen  u.  Leipzig,  1891,  Bd.  xn,  S.  275. 

2  Deutsche  thierarztl  Wchnschr.,  Karlsruhe,  1893,  Bd.  I,  SS.  41,  85  ;  CentraZbl.f. 
Bakteriol.  u.  Parasitenk.,  Jena,  1893,  Bd.  xm,  S.  357 ;  Deutsche  Ztschr.f.  Thiermed., 
Leipzig,  1894,  Bd.  xx,  S.  1. 

3  Rev.  vet.,  Toulouse,  1900,  t.  LVII,  p.  346. 


476  Chapter  XV 

is  made  ten  to  twelve  days  later  and  consists  of  an  inoculation  of  half 
a  cubic  centimetre  of  pure  virus.  This  new  method  had  the  special 
advantage  of  arresting,  almost  immediately,  the  mortality  in  an  in- 
fected piggery  and  of  eliminating  the  chronic  cases  that  are  some- 
times observed  after  the  Pasteurian  vaccinations. 

Leclainche1  has  already  applied  his  method  of  serum  vaccinations 
to  more  than  five  million  pigs  of  all  ages.  "  It  has  been  found  to  be 
constant  in  its  effect  and  absolutely  innocuous,"  and  "not  a  single 
case  of  erysipelas  has  been  met  with  in  pigs  that  had  received  the  two 
vaccines,"  and  Leclainche  hopes  that  his  method  will  soon  come  into 
general  practice,  and  that  it  will  be  utilised  in  all  cases  where  the 
Pasteurian  method  is  found  to  be  insufficient. 

As  the  basis  of  all  the  new  methods  for  vaccinating  pigs  against 
erysipelas  is  the  preparation  of  serums  capable  of  preventing  the 
pathogenic  effect  of  the  bacilli,  the  question  of  the  determination  of 
the  protective  power  of  these  serums  comes  to  be  one  of  considerable 
importance.  At  first  one  was  satisfied  with  certain  approximate 
[499]  estimations,  but  later  the  necessity  was  felt  of  having  a  more  exact 
measurement.  Leclainche  is  persuaded  that  of  all  the  laboratory 
animals  capable  of  being  used  for  these  experiments  the  pigeon  is  the 
only  one  that  can  usefully  fulfil  this  role ;  very  susceptible  to  the 
passage  virus,  it  is  killed  by  the  bacillus  after  a  regular  incubation 
and  invasion  period,  and  the  chronic  form  of  the  erysipelas,  so 
troublesome  in  the  rabbit  and  even  in  the  pig,  is  met  with  in  the 
pigeon  in  very  exceptional  cases  only.  Leclainche  commenced  his 
experiments  by  inoculating  into  the  pectoral  muscles  of  the  pigeon 
mixtures  of  serum  and  virulent  cultures.  The  pigeon  received  1  c.c. 
of  a  culture  of  a  passage  virus  mixed  with  variable  quantities  of 
serum.  The  serum  is  ready  for  use  in  the  vaccination  of  pigs  when 
the  pigeons  resist  the  injection  of  a  mixture  of  £  a  c.c.  of  serum  with 
1  c.c.  of  a  virus  which  kills  the  control  pigeons  in  60  to  72  hours. 

At  the  Frankfort  Institute  of  Experimental  Therapeutics  another 
method  of  testing  devised  by  Marx2  is  used.  In  it  injections,  below 
the  skin  of  a  series  of  grey  mice,  are  made  of  progressively  increasing 
doses  of  the  serum  the  strength  of  which  it  is  desired  to  determine. 
Twenty-four  hours  later  a  virulent  culture  of  the  bacillus  of  swine 
erysipelas  is  introduced  into  the  peritoneal  cavity  of  the  same  mice. 
The  virus  is  so  chosen  that  the  control  mice  die  in  about  72  hours. 

1  Rev.  vet.,  Toulouse,  1901,  t.  LVIII,  p.  149. 

2  Deutsche  thierarztl.  Wchnschr.,  Karlsruhe,  1901,  No.  6. 


Protective  vaccinations  477 

Marx  finds  that  this  method  gives  results  which  are  much  more 
constant  and  exact  than  any  other;  this  opinion  is  confirmed  at 
Hochst,  the  largest  factory  of  serums  in  Germany. 

VIII.  Vaccinations  against  bovine  pleuropneumonia.  This  in- 
fective disease  is  one  of  the  most  dreaded  scourges  of  bovine  animals. 
Very  contagious,  it  has  spread  from  central  Europe  not  only  into  all 
the  other  countries  of  the  European  continent,  but  into  Africa, 
America,  and  almost  every  quarter  of  the  globe.  The  virus  of  this 
disease  was  discovered  in  the  serous  exudation  of  hepatised  lungs 
long  before  the  microbiological  period  of  the  Medical  Sciences  had 
begun. 

Dr  Willems  of  Harselt,  who  made  an  experimental  investigation, 
remarkable  for  the  time  at  which  it  was  carried  out  (more  than  half  a 
century  ago),  demonstrated  at  once  the  great  virulence  of  the  pul- 
monary serous  fluid  ;  he  found  also  that  the  effects  of  the  inoculation 
of  the  virus  varied  much  according  to  the  seat  of  inoculation.  When  [500] 
made  into  the  trunk,  the  neck,  or  the  shoulders,  the  inoculations  are 
usually  fatal ;  at  the  periphery,  the  lower  part  of  the  limbs,  at  the 
extremity  of  the  ears  or  of  the  tail,  the  inoculation  ordinarily  pro- 
duces merely  an  inflammatory  tumefaction  of  small  extent,  which  is 
absorbed  in  a  few  weeks ;  after  this  the  animal  is  refractory  to  the 
natural  disease.  Willems  concluded  from  this  that  we  may  vaccinate 
against  pleuropneumonia  by  inoculating  the  virulent  serous  fluid  of 
the  lung  into  the  tail  Willems'  method  of  inoculation  became  a  part 
of  current  practice  50  years  ago. 

For  the  carrying  out  of  a  large  number  of  vaccinations  it  is 
necessary  to  have  at  one's  disposal  an  adequate  quantity  of  virus ; 
it  was  therefore  to  meet  this  requirement  that  researches  were  first 
carried  out.  The  serous  fluid  was  withdrawn  from  the  hepatised 
lungs  of  animals  that  had  succumbed  to  the  disease  and  was  inocu- 
lated into  normal  Bovidae  as  soon  as  possible,  so  as  to  avoid  con- 
tamination of  the  fluid.  In  fact  this  pulmonary  serous  fluid  often 
contains  foreign  germs  capable  of  multiplying  rapidly  so  that  it  putre- 
fies very  quickly.  Pasteur  showed  that  it  was  possible  to  remedy 
these  drawbacks  by  a  very  simple  method  by  which  he  could  obtain 
a  large  quantity  of  rigorously  pure  virus.  All  that  is  necessary  is  to 
inoculate  a  little  of  the  pleuropneumonic  virus  below  the  skin  of  a 
weaned  calf,  behind  the  shoulder.  At  the  seat  of  inoculation  there 
is  an  abundant  exudation  of  virulent  serous  fluid  into  the  cellular 


478  Chapter  XV 

tissue,  from  which  we  are  enabled  to  collect  large  quantities  of 
pure  virus. 

In  some  countries,  as  in  Germany  and  in  Australia,  institutions 
have  been  founded  for  the  production  by  this  method  of  the  virulent 
serous  fluid  necessary  for  these  inoculations. 

The  virus  should  be  inoculated  into  the  tip  of  the  tail  of  animals 
that  it  is  desired  to  immunise,  because  the  temperature  in  this  situa- 
tion is  relatively  low  and  the  connective  tissue  is  dense  and  not  very 
abundant.  The  inoculation  is  made  with  a  lancet  or  a  Pravaz  syringe. 
The  vaccination  is  generally  borne  well,  in  spite  of  the  reaction 
phenomena  which  are  manifested  about  two  weeks  after  the  intro- 
duction of  the  virus.  At  that  time  a  febrile  condition  is  set  up  and 
a  swelling  manifests  itself  at  the  point  of  inoculation,  which,  however, 
soon  retrogresses  and  then  disappears. 

The  immunity  conferred  by  Willems'  method  is  substantial  and 
lasting  (for  one  or  two  years  and  even  longer) ;  this  explains  its  great 
success  in  the  hands  of  breeders  and  veterinarians.  Accidents  fol- 
[501]  lowing  its  use  are  rare,  and  the  mortality  does  not  exceed  1  per  cent. 

In  spite  of  all  these  advantages  a  new  method  was  still  desirable, 
a  method  which  would  allow  of  the  preparation  of  large  quantities 
of  virus  of  a  suitable  and  uniform  activity  under  conditions  of  irre- 
proachable purity.  Thanks  to  the  discovery  of  the  micro-organism 
of  pleuropneumonia  which  we  owe  to  Nocard  and  Roux1  this  object 
has  been  achieved.  With  the  collaboration  of  Borrel,  Salimbeni,  and 
Dujardin-Beaumetz,  they  succeeded  in  demonstrating  and  isolating 
this  micro-organism,  the  smallest  of  all  known  living  organisms.  The 
first  steps  in  these  researches  were  very  laborious,  but  later  the 
organism  of  pleuropneumonia  was  cultivated  on  fluid  and  solid  media : 
Martin's  broth  (prepared  with  pigs'  stomachs)  or  agar  with  the 
addition  of  a  certain  quantity  (about  5  °/o)  of  fresh  ox  serum.  The 
serum-broth,  sown  with  pure  pneumonic  serous  fluid,  gives  only  a 
moderate  growth,  which  becomes  only  slightly  turbid  and  contains 
micro-organisms  so  small  that  it  is  impossible  to  distinguish  them 
individually.  They  can  be  made  out  only  when  massed  together  in 
irregular  clumps.  The  minuteness  of  this  micro-organism  is  evidenced 
by  the  ease  with  which  it  passes  through  a  Berkefeld  filter,  and  even 
through  certain  Chamberland  candles  (F).  This  feature  enables  us  to 

1  Ann.  de  VInst.  Pasteur,  Paris,  1898,  t.  xn,  p.  240 ;  Cinquanten.  d.  I.  Soc.  d. 
biol.,  Paris,  1899,  p.  440;  Dujardin-Beaumetz,  "Le  microbe  de  la  peripneumonie," 
These  de  Paris,  1900. 


Protective  vaccinations  479 

obtain  the  pure  virus  easily,  a  fact  very  important  in  connection  with 
the  isolation  of  the  micro-organism. 

Once  in  possession  of  pure  cultures  of  the  micro-organism  of  pleuro- 
pneumonia,  Nocard  and  Roux  attempted  to  make  use  of  it  in  practical 
vaccination.  They  showed  that  the  organism  separated  by  them  is 
capable  of  producing  typical  pleuropneumonia  when  it  is  inoculated 
into  the  appropriate  regions  of  the  body  of  bovine  animals.  But 
when  inoculated  subcutaneously  or  into  the  skin  of  the  tail,  it 
produces  merely  a  mild  and  transient  disease  which  confers  an 
immunity  quite  as  effectual  as  that  set  up  by  the  inoculation  of  the 
virulent  serous  fluid.  It  may  be  readily  understood  that,  under  these 
conditions,  pure  cultures  may  be  much  more  serviceably  employed  in 
the  practice  of  vaccination  than  can  Willems'  virus  from  the  fact  that 
it  is  easy  to  obtain  large  quantities  of  absolutely  pure  cultures.  It  is 
easy  to  predict  that  the  new  method  will  soon  replace  the  old  one, 
very  great  as  are  the  services  the  latter  has  rendered  to  agriculture.  [502] 
Up  to  the  present,  vaccinations  with  pure  cultures  have  been  made  in 
several  districts  in  France  with  very  favourable  results.  The  Pasteur 
Institute  and  the  Veterinary  School  at  Alfort  have  already  distributed 
to  veterinary  surgeons  more  than  5,000  vaccinal  doses  of  culture  ;  the 
protective  action  of  these  inoculations  has  been  at  least  equal  to  that 
of  the  inoculations  by  Willems'  method  and  the  resulting  accidents 
have  been  reduced  hi  the  proportion  of  20  to  I1. 

The  serum  of  animals  hyperimmunised  against  pleuropneumonia 
possesses  a  very  distinct  protective  action,  but  too  little  marked  and 
of  too  short  duration  to  be  of  any  use  in  practice ;  it  has  also  a 
curative  action  arresting  the  invading  march  of  a  pleuropneumonic 
congestion ;  but  here  it  is  necessary  to  intervene  early,  before  the 
appearance  of  fever,  and  to  inject  large  quantities  of  serum. 

The  inoculation  of  a  mixture  of  virus  and  serum  produces  no 
congestion ;  but  it  does  not  confer  any  immunity ;  the  animal  remains 
just  as  susceptible  as  the  control  to  the  inoculation  of  the  pure  virus. 

IX.  Vaccinations  against  typhoid  fever.  In  the  preceding 
sections  I  have  treated  more  especially  of  the  vaccination  of  domestic 
animals  against  several  infective  diseases.  The  information  collected 

1  In  1884,  in  the  Department  of  the  Basses- Pyrenees,  the  Willems'  method  of 
inoculation  was  carried  out  on  1354  Bovidae ;  of  this  number  10  died  and  45  lost 
their  tails  completely.  In  1901,  in  the  same  department,  2800  Bovidae  were 
inoculated  with  pure  cultures,  only  1  died  and  9  lost  their  tails. 


480  Chapter  XV 

on  this  subject  is  marked  by  its  great  exactness,  as  it  is  easy  to  apply 
to  animals  the  most  rigorous  experimental  method.  In  the  case  of 
the  human  subject  this  is  not  such  an  easy  matter.  As  it  is  impossible 
to  submit  him  to  experimental  proof  we  are  obliged  to  be  satisfied 
with  observation,  controlled  by  statistical  data.  The  experience  of 
more  than  100  years  has,  however,  been  sufficient  to  demonstrate  the 
great  utility  of  vaccinations  against  small-pox  with  the  virus  of  cow- 
pox  which  is  innocuous  for  the  human  subject.  In  the  case  of  antirabic 
vaccinations  we  have  to  deal  with  injections  into  the  human  subject, 
first  of  weakened  viruses  and  then  of  virulent  viruses.  Here,  how- 
ever, it  is  a  question  of  the  preservation  of  the  already  infected  human 
organism,  which,  very  often,  only  comes  under  treatment  during  the 
incubation  stage  of  rabies.  One  can  readily  understand  the  hesitation 
to  inoculate  even  weakened  viruses  into  the  human  subject,  especially 
[503]  vvhen  we  are  not  dealing  with  altogether  exceptional  cases  such  as 
we  have  in  the  protection  against  rabies.  We  have,  therefore,  but  few 
examples  in  which  the  methods  of  vaccination  by  micro-organisms 
have  been  applied  to  man.  Such  injections  were  first  tried  by  Ferran1 
against  Asiatic  cholera.  Having  succeeded  in  vaccinating  guinea-pigs 
against  experimental  cholera  septicaemia,  the  Spanish  investigator 
attempted  to  inoculate  cholera  vibrios  into  the  subcutaneous  tissue  of 
man,  hoping  thus  to  vaccinate  him  against  true  cholera.  In  this  way 
he  was  able  to  demonstrate  that  the  subcutaneous  injection  of  living 
vibrios  never  sets  up  symptoms  of  cholera.  The  injection  is  followed 
by  a  general  reaction  in  the  form  of  fever,  pains  in  the  back  and 
inflammation  at  the  point  of  inoculation,  in  a  word,  transient  phenomena 
of  little  gravity.  Encouraged  by  these  initial  results  Ferran,  profiting 
by  the  outbreak  of  cholera  in  the  province  of  Valentia,  injected  into 
more  than  20,000  persons  living  cultures  of  Koch's  vibrio.  The  results 
published  by  him  did  not,  however,  furnish  any  real  proof  of  the 
possibility  of  conferring  immunity  against  intestinal  cholera  by  means 
of  subcutaneous  injections.  Later  Haffkine2  modified  Ferran's  primi- 
tive method  somewhat,  and  instead  of  living  vibrios  he  injected 
vibrionic  cultures  killed  by  heat  or  by  antiseptics.  During  the  cholera 
epidemic  of  1892  and  1893  he  tried  the  inoculation  of  these  killed 
vibrios  into  man,  with  the  object  of  vaccinating  against  Asiatic 

"  L'inoculation  preventive  centre  le  cholera  morbus  asiatique"  (translated  from 
the  Spanish),  Paris,  1893. 

2  "Anti-cholera  Inoculations  in  India,"  Indian  Med.  Gaz.,  Calcutta,  Ib95,  .No.  1. 
[Also  Report  to  the  Guv.  of  India,  Calcutta,  1895.] 


Protective  vaccinations  481 

cholera.  Later  he  went  to  Calcutta  in  order  to  try  his  method  on  a  large 
scale.  He  was  there  enabled  to  inoculate  a  great  number  of  persons, 
and  the  statistics  which  he  collected  appeared  to  him  to  be  favourable. 

But  studies  on  the  pathogeuesis  of  Asiatic  cholera  shook  the 
foundations  of  Ferran's  method.  The  injections  of  vibrios,  living  or 
killed,  were  found  quite  capable  of  vaccinating  animals  against 
vibrionic  peritonitis  and  septicaemia,  but  they  appear  to  exert  no 
influence  whatever  against  poisoning  by  the  cholera  toxin.  When 
it  had  been  learnt  how  to  set  up  true  intestinal  cholera  in  young 
rabbits  Ferran's  and  other  similar  methods  of  vaccination  were  used 
in  vain  to  prevent  the  incidence  of  this  disease,  which  is  very  similar 
to  Asiatic  cholera  of  man.  An  experiment1  made  at  the  Pasteur 
Institute  in  Paris  upon  two  persons  vaccinated  by  Haffkine,  showed  [504] 
that  they  were  not  protected  against  the  choleriform  diarrhoea  set 
up  by  the  ingestion  of  the  cholera  vibrios.  A  third  person,  who 
had  never  been  "  vaccinated  "  and  who  served  as  "  control,"  after  the 
ingestion  of  the  same  cholera  culture,  behaved  exactly  as  did  the 
other  two. 

From  all  these  data  the  conclusion  was  drawn  that  in  order  to 
prevent  intestinal  cholera  it  is  necessary  to  use  not  cultures  of 
vibrios,  living  or  dead,  but  antitoxic  serums.  In  fact,  the  majority 
of  young  rabbits  vaccinated  with  these  serums  and  afterwards  sub- 
mitted to  infection  by  the  cholera  virus  through  the  mouth  were 
found  to  be  vaccinated  against  intestinal  cholera.  It  has  not  been 
possible,  as  yet,  to  apply  this  method  to  man,  hence  we  are  unable 
to  give  a  decided  opinion.  Moreover,  as  the  methods  based  on 
Ferran's  principle  have  now  been  abandoned  I  have  not  deemed 
it  necessary  to  devote  a  special  section  to  anticholera  vaccinations. 
I  could  not,  however,  pass  it  by  in  silence,  since  the  attempts  to 
vaccinate  man  against  cholera  have  led  to  the  trial  of  a  similar 
method  against  typhoid  fever. 

Pfeiffer  and  Kolle2  were  the  first  to  inoculate  man  with  typhoid 
coccobacilli  sterilised  by  heat.  They  observed  that  these  injections 
caused  fever,  pretty  violent  pains  in  the  back  accompanied  by  vertigo, 
shivering  and  pain  at  the  point  of  inoculation,  without,  however,  being 
in  any  way  serious  to  health.  At  the  same  time  they  found  that  the 
blood  serum  of  inoculated  persons  acquired  a  very  marked  protective 
power  (for  guinea-pigs  injected  intc  the  peritoneal  cavity  with  lethal 

1  Ann.  de  VInst.  Pasteur,  Paris,  1893,  t.  vn,  p.  579. 

2  Deutsche  med.  Wcknschr.,  Leipzig,  1896,  S.  735. 

B.  31 


482  Chapter  XV 

doses  of  typhoid  cultures)  quite  comparable  to  the  properties 
discovered  by  them  in  the  serum  of  persons  who  had  recovered  from 
typhoid  fever.  Pfeiffer  and  Kolle  believed  that  they  thus  had  a 
proof  of  the  refractory  condition  of  the  individuals  whom  they  had 
submitted  to  these  injections. 

These  experiments  were  continued  by  Wright,  Professor  of 
Pathology  at  Netley,  and  it  is  owing  to  his  unwearied  efforts  that 
science  finds  herself  in  possession  of  very  important  evidence  on  the 
subject  of  protective  inoculations  against  typhoid  fever  in  man. 
According  to  a  verbal  communication  made  to  me  by  Wright,  he 
[505]  has  up  to  the  present  distributed  more  than  300,000  doses  of  his 
antityphoid  vaccine.  This  vaccine  he  prepared  in  the  following  way1. 
The  typhoid  coccobacillus  is  sown  in  carefully  neutralised  broth 
containing  1  %  °f  peptone.  The  flasks  of  culture  are  kept  in  the 
incubator  at  about  37°  C.  for  two  or  three  weeks,  after  which  their 
contents  are  transferred  to  large  flasks  in  order  to  be  submitted  to  a 
temperature  of  60°  0.  This  temperature  is  quite  sufficient  to  kill  all 
the  coccobacilli,  but  for  greater  surety  Wright  added  to  his  cultures 
one-tenth  of  their  volume  of  a  5  °/0  solution  of  carbolic  acid  or  of 
lysol.  The  vaccine,  thus  prepared,  is  examined  as  to  its  toxicity 
for  the  guinea-pig  by  means  of  subcutaneous  injections.  Wright 
injects  into  man  a  dose  of  vaccine  which  is  sufficient  to  kill 
100  grammes  of  guinea-pig  (of  the  weight  of  250  to  300  grammes). 
This  dose  often  amounts  to  half  a  cubic  centimetre,  but  it  may 
have  to  be  increased  to  1  c.c.  and  even  1*5  c.c. 

The  inoculations  are  made  below  the  skin  of  the  flank  or  in 
the  shoulder.  They  are  followed  by  a  rise  of  temperature  which 
commences  as  early  as  two  or  three  hours  after  the  injection.  This 
fever  is  accompanied  by  pains  in  the  back,  nausea,  and  want  of 
appetite.  There  may  even  be  collapse ;  this  led  Wright  to  keep  his 
patient  in  bed  for  some  time  after  the  vaccinal  injection.  Besides 
this  reaction,  there  occurs,  at  the  seat  of  inoculation,  a  swelling  and 
redness,  accompanied  by  pain ;  as  a  rule  all  these  symptoms  have 
disappeared  by  the  end  of  48  hours. 

Wright  convinced  himself  that  the  blood  serum  of  individuals 
treated  by.  his  vaccine,  at  the  end  of  a  certain  time  acquires  the 
property  of  agglutinating  typhoid  coccobacilli  in  a  variable,  but 
usually  very  marked  degree.  He  even  thought  that  this  property 

1  Wright  and  Leishman,  Brit.  Med.  Journ.,  London,  1900,  Vol.  I,  p.  122;  [Wright, 
"A  short  treatise  on  anti-typhoid  inoculation,"  London,  1904]. 


Protective  vaccinations  483 

might  up  to  a  certain  point  serve  as  the  measure  of  the  immunity 
acquired  against  typhoid  fever.  His  own  researches,  however,  showed 
him  that  this  supposition  could  not  be  maintained,  and  that  the 
agglutinative  power,  varying  greatly  in  strength,  might  sometimes  be 
absent  where  the  immunity  could  not  be  denied.  On  the  other  hand, 
he  clearly  showed,  especially  by  the  experiments  with  serum  collected 
at  the  period  which  precedes  the  relapses,  that  the  agglutinative 
property  might  be  highly  developed,  in  spite  of  the  absence  of  im- 
munity. Wright  then  set  himself  to  study  the  bactericidal  property 
of  the  serum  of  individuals  who  had  been  injected  with  his  vaccine.  [506] 
He  devised  a  very  ingenious  method  of  gaining  with  a  minimum  loss 
of  time  some  idea  of  the  fluctuations  of  this  power  of  the  body  fluids 
to  kill  the  typhoid  coccobacillus.  In  the  first  place  he  demonstrated 
that  the  bactericidal  property  is  not  at  all  parallel  to  the  agglutinative 
power,  and  this  has  further  confirmed  him  in  his  opinion  that  there 
may  be  no  direct  relation  between  it  and  acquired  immunity.  He  has 
found  further  that  the  power  of  the  blood  serum  to  destroy  the 
typhoid  coccobacillus  is  very  variable  in  persons  vaccinated  by  his 
method.  After  injections  of  large  quantities  of  these  killed  bacilli 
this  power  may  even  be  diminished  for  a  very  long  period.  On  the 
other  hand,  medium  or  small  doses  of  the  vaccine  first  set  up  a 
negative  stage,  during  which  the  bactericidal  property  is  very  feeble, 
and  later  they  bring  about  an  increase  of  this  property,  often  very 
marked.  Wright  does  not  think  that  the  bactericidal  power  can 
serve  as  the  measure  of  the  immunity  acquired  by  the  vaccinated 
individuals,  but  he  hopes  that  some  day  a  method  may  be  found 
suitable  for  the  examination  of  the  blood  which  will  give  us  informa- 
tion as  to  the  degree  of  immunity  conferred  by  the  antityphoid 
vaccination.  For  the  present  the  only  basis  upon  which  we  can  form 
any  opinion  on  this  subject  is  furnished  by  statistics.  Now  we  know 
that  it  is  often  very  difficult  to  collect  data  that  are  sufficiently  exact 
Hence  during  the  war  in  South  Africa,  where  one-fifth  of  the  English 
troops,  that  is  to  say  about  50,000  persons,  were  submitted  to 
vaccinations  by  Wright's  method,  it  is  only  in  certain  cases  that  the 
statistical  information  can  be  utilised.  Many  of  the  patients  attacked 
by  slight  fevers  are  omitted  from  the  statistics,  because  from  the 
absence  of  a  precise  diagnosis  it  is  not  known  whether  they  should 
come  under  the  category  of  typhoid  patients  or  not.  In  other  cases 
the  secondary  complications  divert  the  attention  of  the  doctors  and 
prevent  the  registration  of  a  proper  diagnosis. 

31—2 


484  Chapter  XV 

Of  the  data  collected  amongst  the  English  troops  in  South  Africa, 
Wright  considers  that  those  which  were  collected  during  the  siege 
of  Ladysmith  were  the  most  exact,  on  account  of  the  facility  with 
which  it  was  possible  to  study  and  register  all  the  cases  of  typhoid 
fever  under  these  conditions  of  complete  isolation.  Now  it  has  been 
recognised  that,  amongst  the  vaccinated  soldiers  and  officers,  there 
occurred  scarcely  one-eighth  as  many  cases  of  typhoid  fever  as 
occurred  amongst  the  un vaccinated  (1,499  cases  in  10,529  unvac- 
cinated, and  35  cases  in  1,705  vaccinated).  The  mortality  amongst 
[507]  the  vaccinated  was  also  very  much  lower.  The  difference  to  the 
credit  of  the  vaccinations  should  in  reality  be  even  greater,  for 
amongst  the  unvaccinated  are  counted  many  persons  who  having 
already  had  an  attack  of  typhoid  fever  were  not  submitted  to 
vaccination. 

The  testimony  of  the  majority  of  the  medical  men  who  followed 
the  results  of  Wright's  method  closely  is  also  favourable  to  the 
vaccinations.  Thus  Henry  Cayley1  reports  that  the  staff  of  a  Scotch 
Hospital  of  the  Red  Cross,  almost  all  of  whom  (57  persons  out  of  61) 
had  received  two  vaccinal  inoculations,  escaped  typhoid  fever,  in 
spite  of  the  numerous  opportunities  afforded  for  the  contraction 
of  the  disease.  This  very  favourable  example  is  also  instructive 
in  that  it  testifies  to  the  value  of  two  consecutive  vaccinations.  In 
many  other  cases  where  one  has  had  to  be  satisfied  with  a  single 
protective  inoculation  the  results  were  less  brilliant.  According  to 
Howard  Tooth,  who  made  his  observations  at  Bloenifontein,  the 
vaccinations  according  to  Wright's  method  must  be  regarded  as 
very  useful. 

Outside  South  Africa  this  method  has  been  employed  on  a 
fairly  large  number  of  persons  in  British  India,  in  Egypt,  and 
in  Cyprus.  According  to  the  earlier  statements  from  India  the 
incidence  amongst  the  vaccinated  persons  was  one-third  that  of 
the  unvaccinated.  The  most  recent  statistics2  show  still  more 
favourable  results.  Thus  at  Meerut  the  incidence  amongst  vaccinated 
persons  from  Oct.  1899  to  Oct.  1900  was  one-eleventh  that  of  the  un- 
vaccinated (2  cases  of  typhoid  fever  in  360  vaccinated,  and  11  cases 
of  the  same  disease  in  179  unvaccinated) :  the  mortality  (one  case 
amongst  the  former,  six  amongst  the  latter)  was  less  than  one-twelfth 
that  of  the  unvaccinated. 

1  Brit.  Med.  Journ.,  London,  1901,  Vol.  I,  p.  84. 

2  Lancet,  London,  1901,  Vol.  I,  p.  399. 


Protective  vaccinations  485 

In  Egypt  and  in  Cyprus  according  to  the  statistics  communicated 
to  Dr  Wright1  by  Col.  Fawcett  these  vaccinations  have  given  even 
better  results.  In  2,669  unvaccinated  persons  there  occurred  68  cases 
of  typhoid  fever  with  10  deaths,  whilst  amongst  the  720  vaccinated 
there  was  only  a  single  case  of  this  disease,  this  single  case  suc- 
cumbing. Here,  however,  we  have  to  do  with  a  patient  who  must 
have  received  the  vaccinal  inoculation  during  the  period  of  incubation, 
the  disease  breaking  out  soon  after  the  vaccination.  This  would 
represent  in  all  the  cases  a  morbidity  only  one-seventeenth  as  intense 
amongst  the  vaccinated. 

A  few  isolated  voices  only  have  not  pronounced  in  favour  of  the  [508] 
antityphoid  vaccinations  and  their  opinion  is  formulated  in  a  very 
undecided  fashion.  Amongst  the  most  important  of  these  adver- 
saries, if  indeed  we  may  term  them  such,  must  be  cited  Washbourn2, 
on  account  of  his  experience  in  microbiology.  Attached  as  a  doctor 
to  the  Yeomanry  Hospital  at  Deelfontein  in  South  Africa,  he 
witnessed  many  cases  of  typhoid  fever  and  was  greatly  struck  by  the 
death  of  two  persons  amongst  the  vaccinated  patients.  But  he 
himself  confesses  that  it  is  as  yet  premature  to  judge  Wright's 
method,  and  in  support  of  his  sceptical  attitude  does  not  offer  any 
other  satisfactory  observation. 

Outside  the  English  colonies  vaccinations  against  typhoid  fever 
have  been  tried  in  Russia  by  Wyssoko witch3.  He  inoculated 
235  soldiers  of  a  regiment  encamped  at  Kiew,  amongst  whom  an 
epidemic  of  typhoid  fever  had  broken  out.  The  vaccinations  were 
carried  out  by  means  of  cultures  killed  with  carbolic  acid.  We  are 
unable  to  judge  of  the  efficacy  of  the  method  because  the  number 
of  persons  vaccinated  was  too  small  and  the  epidemic  too  limited. 
It  may  be  noted,  however,  that  amongst  these  individuals  not  one 
took  typhoid  fever,  whilst  amongst  the  unvaccinated  three  cases 
of  the  disease  were  registered. 

The  antityphoid  vaccinations  have  as  yet  only  a  very  short  history, 
and  it  is,  perhaps,  premature  to  express  any  decided  opinion  on  the 
matter.  We  may,  however,  consider  the  results  already  obtained  as 
offering  encouragement  to  continue  our  experiments.  Everything, 
indeed,  tends  to  a  recognition  of  the  utility  of  vaccinations  by  means 
of  killed  typhoid  cultures.  The  statistics  are  as  a  rule  good  ;  the 

1  Lancet,  London,  1901,  Vol.  i,  p.  1272. 

2  Brit.  Med.  Journ.,  London,  1900,  Vol.  i,  p.  1456. 

3  Gaz.  din.  de  Botkine,  St  Petersb.,  1899,  p.  1911  (in  Russian). 


486  Chapter  XV 

danger  from  the  protective  inoculation  is  nil  or  quite  trifling.  With 
the  exception  of  the  discomfort  of  which  we  have  spoken  and  which 
is  transitory,  no  untoward  result  has  ever  been  observed. 

To  all  this  must.be  added  the  fact  that  from  the  point  of  view 
of  the  pathogenesis  of  typhoid  fever,  all  the  probabilities  point  in 
favour  of  the  vaccinations.  Whilst  in  Asiatic  cholera  we  have  to 
deal  with  an  intoxication,  from  the  alimentary  canal,  an  intoxication 
set  up  by  vibrionic  products,  against  which  the  subcutaneous  inocu- 
lation of  micro-organisms  can  not  be  effective,  in  typhoid  fever  we 
have  to  do  with  a  real  infection.  The  micro-organism,  although  de- 
veloped at  first  in  the  small  intestine,  becomes  generalised  throughout 
the  system.  Thanks  to  improved  methods  it  can  always,  or  almost 
[509]  always,  be  found  in  the  blood  of  the  patient,  and  its  constant  locali- 
sation in  the  spleen  furnishes  a  real  evidence  of  this.  Under  these 
conditions  it  is  quite  natural  to  suppose  that  everything  which  is  able 
to  prevent  the  penetration  of  the  typhoid  coccobacillus  into  the  blood 
and  the  internal  organs  ought  at  the  same  time  to  contribute  to  the 
protection  of  the  individual. 

We  are  fully  aware  that  science  has  not  yet  said  its  final  word 
upon  this  question.  We  are  coming  more  and  more  to  the  conclusion 
that  it  is  necessary  to  make  two  injections  instead  of  one.  It  is 
possible  that  we  may  have  recourse  to  certain  improvements  of  the 
method  by  combining  with  it  the  injections  of  antityphoid  serums  as 
a  protective  measure.  The  near  future  will  doubtless  bring  us  the 
solution  of  these  very  important  questions. 

X.  Vaccinations  against  human  plague.  Plague,  which  for  so 
long  was  looked  upon  as  the  greatest  scourge  of  humanity,  has  until 
recently  remained  almost  unknown  from  the  scientific  point  of  view. 
But  from  the  moment  that  it  became  possible  to  apply  to  its  study 
the  immense  advances  realised  by  microbiology  the  thick  veil  which 
had  hidden  its  nature  fell  at  a  single  stroke  and  science  found  itself 
in  possession  of  effective  means  of  fighting  against  it.  Amongst  these 
means  one  of  the  most  important  is  protective  vaccination. 

When  the  last  pandemic  of  plague  broke  out  in  Bombay  and  in 
the  East  Indies  in  general,  Haffkine  was  there  engaged  in  applying 
his  method  of  vaccination  against  Asiatic  cholera  of  which  we  have 
spoken  in  the  preceding  section.  Well  acquainted  with  the  results 
of  the  bacteriological  researches  made  on  bubonic  plague  by 
Kitasato,  and  especially  by  Yersin,  he,  in  1896,  began  to  study  this 


Protective  vaccinations  487 

disease.  After  the  discovery  made  by  Yersin,  Borrel,  and  Calmette1, 
who  showed  that  animals  susceptible  to  human  plague  could  be 
easily  vaccinated  against  the  micro-organism  which  gives  rise  to  it, 
Haffkine2  endeavoured  to  find  a  practical  method  for  the  vaccination 
of  man.  He  set  up  a  laboratory  at  Bombay  and,  after  some 
preliminary  experiments  on  rabbits,  he  commenced  to  inject  human 
beings  with  pure  cultures  of  the  plague  coccobacillus.  From 
1897  up  to  the  present  he  was  able  to  vaccinate  a  very  large  [510] 
number  of  individuals,  and  the  results  obtained  have  encouraged 
him  to  continue  the  application  of  his  method.  The  principle 
of  this  method  is  that  which  had  guided  him  in  the  preparation 
of  anticholera  vaccines  and  which  is  used  for  the  vaccines  against 
typhoid  fever.  It  consists  in  the  employment  of  pure  cultures 
of  the  specific  organism  killed  by  heat.  The  cultures  are  grown 
in  large  flasks  containing  peptonised  broth  and  sown  with  a  small 
quantity  of  the  plague  coccobacilli.  A  little  sterile  butter  or 
cocoanut  oil  is  poured  on  the  surface  of  the  fluid.  Under  these 
conditions  the  organism  grows  abundantly  and  produces  growths 
which  hang  down  into  the  fluid,  reminding  us  of  the  stalactites  in 
a  grotto.  This  mode  of  development  forms  one  of  the  most  typical 
characters  of  the  micro-organism  of  human  plague.  The  culture 
flasks  are  kept  at  a  temperature  of  about  30°  C.  for  five  to  six  weeks, 
at  the  end  of  which  period  a  large  number  of  the  bodies  of  the  micro- 
organisms have  fallen  to  the  bottom  of  the  flask,  allowing  much  of 
their  toxic  contents  to  escape.  The  fatty  layer  on  the  surface  favours 
a  surface  development  ot  the  coccobacilli,  the  number  of  micro-organ- 
isms in  a  flask  being  thus  greatly  increased. 

After  growing  for  35  to  42  days  under  these  conditions  the 
cultures  are  heated  at  65° — 70°  C.  for  from  one  to  three  hours  with 
the  object  of  killing  all  the  micro-organisms  and  so  rendering  their 
injection  innocuous.  To  make  sure  of  the  effectiveness  of  this 
heating  care  is  taken  to  remove  a  small  portion  of  the  fluid  and  to 
sow  it  in  a  suitable  medium.  Should  this  medium  remain  sterile  the 
vaccine  may  be  used.  Into  adult  men  it  is  injected  in  a  dose  of  3  c.c., 
whilst  women,  children,  and  adolescents  receive  2 — 2*5  c.c.,  into  the 
subcutaneous  tissue. 

Some  hours  after  the  injection  of  the  vaccine  the  temperature 

1  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  589. 

2  Brit.  Med.  Journ.,  London,  1897,  Vol.  i,  p.  1461 ;  Indian  Med.  Gaz.,  Calcutta, 
1897,  Vol.  xxxii,  p.  201. 


488  Chapter  XV 

rises  above  normal,  reaching  38'5°  to  39°  C.,  and  sometimes  even 
40°— 40' 5°  C.  This  febrile  condition  lasts  from  15  to  48  hours.  It  is 
soon  accompanied  by  pain,  redness,  and  swelling  at  the  point  of 
inoculation.  These  symptoms  persist  for  from  three  to  five  days. 
The  malaise  which  follows  the  vaccinations  is  sometimes  very  uncom- 
fortable or  even  painful,  but  never  serious.  Only  in  exceptional 
cases  is  the  formation  of  abscesses  observed,  and  this  is  due,  undoubt- 
edly, to  contamination  of  the  vaccines  by  foreign  micro-organisms. 
The  English  Commission  sent  to  India  to  study  plague  found  other 
micro-organisms  than  the  plague  coccobacilli  fairly  frequently  in  the 
[511]  vaccine  culture  flasks,  but,  with  very  rare  exceptions,  these  micro- 
organisms were  found  to  be  innocuous.  By  rigorously  following  the 
rules  to  be  observed  in  making  pure  cultures  it  should  not  be  diffi- 
cult to  avoid  this  complication. 

HaiFMne  used  every  effort  to  induce  his  patients  to  be  vaccinated 
a  second  time,  being  justly  persuaded  that  two  injections  are  capable 
of  ensuring  a  more  certain  and  more  stable  immunity  than  is  a  single 
injection. 

From  what  moment  immunity  may  be  considered  to  be  acquired 
has  been  a  matter  for  great  discussion.  From  very  numerous  experi- 
ments upon  animals  of  various  species,  as  well  as  many  observations 
on  man,  it  is  now  agreed  that  a  period  of  several  days  (5 — 8)  from 
the  injection  of  the  vaccine  is  required  before  immunity  is  manifested. 
It  is  for  this  reason  that  cases  of  plague  which  have  broken  out 
before  this  period  has  elapsed  cannot  be  looked  upon  as  contra- 
indicating  the  efficacy  of  the  method. 

A  large  amount  of  evidence,  coming  from  persons  who  have  made 
their  observations  on  the  spot,  is  almost  unanimous  in  endorsing  the 
fact  that  Haffkine's  vaccination  protects  man  against  plague.  It  is 
often  difficult  to  compile  exact  statistics  in  surroundings  where  so 
many  factors  contribute  to  deceive  even  the  careful  observer.  In 
spite  of  this  a  certain  amount  of  evidence  has  been  collected  which 
may  be  accepted  as  affording  us  fairly  satisfactory  information.  One 
of  the  best  groups  of  statistics  was  that  collected  at  Damaun, 
a  Portuguese  possession  in  India,  into  which  plague  was  imported  from 
Bombay  in  1897,  and  where  a  large  number  of  vaccinations  were 
carried  out.  From  the  report  of  Haffkine  and  Lyons1,  in  a  popula- 
tion of  8230  persons,  rather  more  than  one-fourth  (2197)  were 
vaccinated,  the  greater  majority  (6033)  remaining  uninoculated. 

1  "Joint  Report  on  the  Epidemic  of  Plague  in  Lower  Damaun,"  Bombay,  1897. 


Protective  vaccinations  489 

Amongst  the  former  only  36  died  from  plague,  which  corresponds 
to  1*6  per  cent. ;  whilst  amongst  the  unvaccinated  persons  the  disease 
carried  off  1482  persons  or  24'6  per  cent.  Vaccination,  therefore, 
according  to  these  statistics,  must  have  brought  down  the  mortality 
to  one-fifteenth.  The  German  Commission1,  two  members  of  which, 
Koch  and  Gaffky,  went  to  Damaun  to  be  present  at  the  vaccinations 
and  to  observe  their  efficacy,  pronounced  in  favour  of  Haffkine's 
method.  The  English  Commission2  made  reservations  and  criticised 
the  statistics  of  Haffkine  and  Lyons  (who  amongst  others  attribute 
all  the  cases  of  deaths  that  occurred  amongst  the  unvaccinated  to  [512] 
plague),  but  in  the  end  this  Commission  also  recognised  the  utility 
of  the  vaccinations  at  Damaun. 

The  data  collected  with  regard  to  the  vaccinations  at  Undhera, 
Hubli,  and  several  other  places  in  British  India  confirm  the  results 
obtained  at  Damaun.  The  statistics  collected  at  these  localities 
are  certainly  open  to  criticism,  but  the  result  as  a  whole  is 
none  the  less  encouraging  as  regards  this  method  of  vaccination. 
According  to  the  conclusions  of  the  English  Commission  the  "  inocu- 
lations had  a  considerable  effect  in  warding  off  plague  attacks  from 
the  inoculated.... The  protection  afforded  by  inoculation  seems,  how- 
ever, never  to  be  absolute3."  We  do  not,  as  yet,  know  the  duration 
of  the  immunity  produced  by  Haffkine's  vaccinations  ;  it  cannot  be 
very  long  to  judge  from  the  experiments  on  animals,  but  it  may  last 
for  several  weeks,  probably  even  for  months. 

The  vaccinations  by  killed  cultures  may  be  especially  useful  when 
it  is  a  question  of  limiting  the  extension  of  an  epidemic  that  is 
already  established.  The  ease  with  which  the  vaccine  can  be  prepared 
renders  it  possible  to  obtain  very  large  quantities  of  it  in  a  short 
time,  with  which  it  is  possible  to  immunise  the  entire  population  of 
towns  or  districts.  But,  as  the  immunity  by  this  method  requires 
several  days  for  its  development  and  as  the  injections  of  micro- 
organisms, even  when  killed,  may  be  very  injurious  during  the 
incubation  period  of  plague  or  immediately  before  the  infection,  it  is 
necessary  to  limit  the  vaccinations  to  persons  who  are  not  in  intimate 
contact  with  the  sick,  or  who  are,  from  the  beginning,  exposed  to 
infection4. 

1  Arb.  a.  d.  K.  Gsndhtsamte,  Berlin,  1899,  Bd.  xvi,  S.  331. 

2  "  Report  of  the  Indian  Plague  Commission,"  London,  1901,  VoL  v,  Chapter  iv. 

3  Ibid.  Chapter  iv,  p.  81. 

4  See  Calmette,  "  Rapport  sur  les  vaccinations  centre  la  peste,"  Compt.  rend.  d. 
X  Congr.  internal.  d''kyg.  de  Paris,  1900. 


490  Chapter  XV 

Lustig  and  Galeotti1  have  described  another  method  of  preparing 
antiplague  vaccine  which  can  be  utilised  where  it  is  of  importance  to 
obtain  a  large  quantity  of  vaccine  in  a  very  short  time.  Instead  of 
allowing  the  cultures  to  grow  for  five  or  six  weeks  as  required  by 
Haffkine's  method,  the  Italian  observers  make  use  of  cultures  on  agar 
which  have  grown  for  two  days  only.  The  micro-organisms,  removed 
from  the  surface  of  the  agar,  are  treated  with  a  weak  solution  of 
potash  (0*75  °/0 — 1  %)  which  dissolves  the  bodies  of  the  coccobacillL 
[513]  This  phenomenon  has  sometimes  occurred  by  the  end  of  twenty 
minutes,  but  it  often  requires  an  hour  or  more.  The  contact  of  the 
micro-organisms  with  the  alkali  must  never  exceed  three  hours. 
The  viscous  mass  thus  obtained  is  then  treated  with  acetic  acid,  when 
a  precipitate  is  thrown  down.  This  precipitate,  after  being  washed,  is 
used  for  the  vaccinations.  When  injected  in  large  quantities  into 
animals,  Lustig  and  Galeotti's  product  sets  up  necrosis,  but  a  weak 
dose  is  well  borne  and  confers  immunity  against  plague.  In  man  it 
is  sufficient  to  inject  two  or  three  milligrammes  of  this  substance 
diluted  with  water.  The  vaccinal  nuclein  of  the  Italian  observers  has 
been  but  little  employed  for  the  immunisation  of  man  in  India,  but 
it  is  largely  used  in  this  country  for  the  inoculation  of  horses  from 
which  to  obtain  an  antiplague  serum. 

The  serotherapeutics  against  human  plague  were  inaugurated  by 
the  researches  of  Yersin,  Borrel,  and  Calmette  (l.c.\  who  demon- 
strated that  animals  susceptible  to  the  plague  bacillus  can  be 
vaccinated  and  even  cured  of  experimental  plague.  The  preparation 
of  antiplague  serum  has  since  been  energetically  pursued  under 
Roux's  direction  at  the  Pasteur  Institute.  After  several  trials, 
some  of  which  were  very  encouraging,  others,  on  the  contrary, 
somewhat  unfavourable,  they  succeeded  in  obtaining  a  serum  which 
is  capable  of  curing  plague  after  it  has  broken  out  and  has  become 
grave.  As  in  this  treatise  we  intentionally  leave  aside  everything 
connected  with  healing  we  shall  speak  only  of  the  antiplague  serum 
as  a  protective  agent. 

Whilst  vaccinations  by  killed  plague  cultures  have  been  practised 
principally  in  the  East  Indies,  the  immunisation  with  antiplague  serum 
has  been  employed  in  Europe,  especially  at  the  time  of  the  epidemics 
of  Oporto  in  1899  and  of  Glasgow  in  1900.  In  all  these  cases  use  was 
made  of  the  serum  from  the  Pasteur  Institute,  up  to  the  present  the 
most  active  of  all  those  prepared.  It  is  a  serum  obtained  from 
1  Deutsche  med.  Wchnschr.,  Leipzig,  1897,  SS.  227,  289- 


Protective  vaccinations  491 

orses  treated  for  a  long  period  with  cultures  of  the  plague  bacillus 
nd  with  the  toxin  of  the  same  organism.  Treatment  is  begun  by 
njecting  plague  coccobacilli  killed  by  heat  (70°  C.).  These  injections 
re  made  into  the  veins,  with  the  object  of  avoiding  the  local  lesions 
~hich  are  observed  after  the  subcutaneous  introduction  of  micro- 
rganisms.  When  the  horses  have  been  rendered  refractory  by  this 
reatment  with  dead  micro-organisms,  the  next  step  is  to  inject  (also 
ato  the  veins)  small  quantities  of  living  cultures.  The  doses  of  these 
ultures  are  gradually  increased,  and  end  by  conferring  upon  the  animal  [514] 

very  strong  immunity,  which  is  strengthened  by  injections  of 
roducts  of  cultures  passed  through  a  Chamberland  filter. 

Calmette  and  Salimbeni1  injected  prophylactically  more  than 
00  persons  menaced  by  plague  at  Oporto.  These  comprised  the 
octors  and  the  staffs  of  the  laboratories  of  hygiene  and  of  the 
isinfection  services,  the  firemen  who  removed  the  sick  persons  and 
he  dead,  the  families  of  those  who  were  attacked,  the  members  of 
he  French  colony,  etc.  Into  each  person  5  c.c.  of  serum  was  injected 

ow  the  skin  of  the  abdomen.  These  vaccinations  in  some  cases 
aused  nettle-rash,  eruptions  similar  to  those  so  often  observed  after 
he  injection  of  the  other  kinds  of  serums.  Of  the  total  number  in- 
ected  two  persons  contracted  plague :  the  unfortunate  Doctor  Camera 
Pestana  and  his  assistant.  The  former  succumbed  to  the  disease,  but 
he  second  only  contracted  a  very  mild  form  of  it.  The  study  of 
hese  600  cases,  as  well  as  of  experiments  on  animals,  demonstrated 
hat  the  immunity  conferred  by  the  antiplague  serum  is  set  up 
mmediately  after  its  injection  but  is  not  of  long  duration.  It  is 
>robable  that  it  lasts  for  8  or  10  days,  or  at  furthest  a  fortnight  only. 

Similar  results  were  obtained  at  Glasgow.  Van  Ermengem2,  who 
las  published  a  report  on  the  epidemic  in  this  town,  mentions  that 
more  than  70  persons  in  good  health  were  inoculated  with  the  serum  ; 
?ach  one  received  10  c.c.  beneath  the  skin  of  the  belly.  Of  these 
70  persons  one  was  attacked  with  a  fairly  mild  plague  8  days  after 
he  vaccination,  and  another,  a  housekeeper,  was  attacked,  9  days  after 
he  injection,  with  a  congestion  of  the  cervical  glands  induced  by  the 
plague  bacillus.  Both  cases  recovered.  All  the  other  vaccinated 
>ersons,  in  spite  of  constant  exposure  to  the  plague  infection, 
remained  unaffected.  Van  Ermengem  was  of  opinion  that  the  two 


1  Ann.  de  VInst.  Pasteur,  Paris,  1899,  t.  xin,  p.  902. 

2  Bull.  Acad.  roy.  de  med.  de  Belg.,  Bruxelles,  1900,  27  Octobre. 


492  Chapter  XV 

persons  treated  with  the  serum  were  already  infected  when  they 
were  vaccinated. 

The  Belgian  observer  points  out,  further,  the  frequency  of  secon- 
dary accidents  which  were  produced  in  the  persons  vaccinated  at 
Glasgow.  Van  Ermengem  himself  went  through  the  ordeal  after 
being  injected  with  10  c.c.  of  serum  as  a  protective  measure  and  this 
gave  occasion  to  several  critics  to  attack  the  Pasteur  Institute.  This 
is  how  Van  Ermengem  himself  puts  the  matter.  "  The  accidents  after 
[515]  the  immunising  injections... were  very  numerous,  they  were  observed 
33  times  in  72  cases.  Sometimes  they  were  even  fairly  serious,  to  the 
point  of  causing  great  suffering  to  the  patient  and  of  disquieting 
those  around  them.  We  could  describe  them  from  thorough  know- 
ledge, since  we  experienced  them,  but  they  scarcely  differ  from  those 
which  are  observed  from  time  to  time  after  the  injection  of  anti- 
diphtheria  serum,  and,  like  them,  they  disappear  without  leaving  the 
least  trace"  (Lc.  p.  18). 

In  spite  of  these  accidents  and  the  necessity  of  renewing  fre- 
quently (every  ten  or  fifteen  days)  the  protective  injections  of  serum, 
their  use  is  quite  advisable  in  certain  circumstances.  They  may  render 
great  service  on  board  infected  vessels  or  in  lazarettos  (as  in  the  case 
which  occurred  at  Frioul  after  the  arrival  at  Marseilles  of  Arab 
stokers  suffering  from  plague),  in  docks,  warehouses,  and  stores  where 
contaminated  merchandise  is  found.  They  should  also  be  employed 
to  vaccinate  those  coming  into  immediate  contact  with  plague  cases  in 
hospitals  and  in  private  houses.  In  a  word,  vaccinations  by  serum, 
owing  to  their  power  of  conferring  a  very  rapid  immunity,  should  be 
practised  wherever  there  is  more  or  less  immediate  and  imminent 
danger.  Under  these  conditions  they  are  of  very  great  service  in 
localising  the  disease. 

The  methods  of  vaccination  against  plague  that  have  been 
employed  up  to  the  present  may  undoubtedly  be  improved.  Calmette 
and  Salimbeni  (I.e.)  have  already  published  the  results  of  experi- 
ments on  animals  undertaken  with  the  object  of  studying  the  effect 
of  a  combined  method  of  vaccination  with  antiplague  serum  and 
killed  cultures  of  the  plague  bacillus.  But  even  in  their  present  form 
the  methods  used  for  protecting  individuals  against  this  disease  de- 
serve to  be  regarded  as  conferring  great  benefits  on  humanity. 

XL  Vaccinations  against  tetanus.  Tetanus  unlike  plague  is 
not  a  contagious  disease,  nor  is  it  capable  of  becoming  epidemic. 


Protective  vaccinations  493 

It  constitutes,  however,  a  very  formidable  disease  against  which  all 
I  therapeutic  methods  have  only  a  very  limited  effect.  This  is  a  further 
(reason  for  drawing  the  whole  attention  of  medical  and  veterinary 
imen  to  the  prevention  of  tetanus  by  vaccinal  injections.  Tetanus  is 
a  disease  in  which  the  intoxication  plays  an  altogether  dominant  part. 
The  tetanus  bacilli  do  not  develop,  at  the  point  where  they  are 
introduced  into  the  body,  unless  favoured  by  auxiliary  conditions,  [516] 
such  as  the  multiplication  of  other  micro-organisms.  Even  then  the 
organism  of  tetanus  reproduces  itself  with  difficulty,  and  without 
becoming  generalised  throughout  the  body.  The  poison  which  it 
secretes  is  however  sufficient  to  produce  a  very  grave  intoxication, 
ending  most  frequently  in  death.  In  certain  countries  tetanus,  as 
a  sequel  to  various  wounds,  is  very  frequently  met  with  in  man  and 
in  certain  domestic  animals,  such  as  the  horse,  donkey,  pig,  etc. 

It  is  only  since  the  discovery  by  von  Behring  and  Kitasato  of  an 
effective  method  of  immunisation  against  tetanus  that  it  has  been 
possible  to  consider  the  practical  application  of  antitetanus  vacci- 
nations. These  observers  demonstrated  that  the  tetanus  poison, 
when  treated  with  trichloride  of  iodine,  had  its  toxic  action  weakened 
and  was  transformed  into  an  effective  vaccine.  Roux  and  Vaillard 
found  that  the  addition  of  LugoPs  iodo-iodurated  solution  to  the 
tetanus  poison  renders  it  capable  of  vaccinating  all  kinds  of  suscep- 
tible animals.  It  was  shown  later,  that  even  with  modified  active 
tetanus  toxin,  we  can  still  obtain  good  results  when  care  is  taken 
to  inject  the  poison  with  great  circumspection. 

But  it  is  not  these  vaccines  obtained  from  tetanus  cultures  that 
have  come  to  be  used  in  practice.  The  best  results  are  obtained  by 
the  use  of  antitetanus  serums.  After  von  Behring  and  Kitasato's 
discovery  of  the  power  of  the  serum  of  animals  immunised  against 
tetanus  to  neutralise  the  action  of  the  tetanus  poison,  very  numerous 
experiments  were  made  on  the  same  subject.  It  has  now  become 
possible  by  treating  horses  with  large  quantities  of  tetanus  toxin  to 
obtain  specific  serums  of  extraordinary  activity  Thus  several  serums 
are  capable  of  preserving  mice  against  a  lethal  dose  of  tetanus  poison 
if  we  inject  into  them  a  quantity  of  serum  equal  to  the  one-thousand- 
millionth  of  their  weight. 

Serums  of  this  strength  protect  domestic  animals  against  tetanus. 
We  know  that  many  operations  on  horses,  sheep,  goats,  pigs,  and 
other  mammals  are  very  often  followed  by  a  tetanus  which  is  usually 
fatal.  Castration,  amputation  of  the  tail,  the  ablation  of  proud  flesh 


494  Chapter  XV 

or  tumours,  the  operation  for  cryptorchitis  or  hernias,  etc.  are  often 
complicated  by  tetanus.  Moreover,  tetanus  may  frequently  appear 
in  horses  that  have  received  wounds  in  the  foot  or  in  the  lower 
parts  of  the  limbs,  "Clous  de  rue,"  farrier's  punctures,  wire-heels, 
blows,  etc. 

[517]  With  the  object  of  remedying  this  state  of  things  Nocard1  distri- 
buted to  veterinarians  about  70  litres  of  antitetanus  serum  to  be 
employed  for  protective  purposes.  The  majority  of  the  animals 
treated  (horses,  donkeys,  mules,  bulls,  rams,  lambs,  and  pigs)  received 
two  injections  of  serum  at  an  interval  of  10 — 12  days,  20  c.c.  for 
large  animals  and  6 — 10c.c.  for  sheep  and  pigs.  Of  3088  animals 
which  received  the  first  injection  of  serum  immediately  after  the 
operation  not  a  single  one  contracted  tetanus.  Of  400  animals  which 
received  the  first  injection  at  a  later  period,  1 — 4  days  and  more 
after  the  accidental  wound  of  which  they  had  been  the  victims, 
one  horse  only,  treated  five  days  after  the  accident  (farrier's  punc- 
ture), was  seized  with  mild  tetanus,  but  it  soon  recovered.  In  the 
same  localities  where  the  results  of  the  vaccination  were  so  brilliant, 
314  cases  of  grave  and  fatal  tetanus  occurred  amongst  animals  operated 
upon  or  injured  that  were  not  submitted  to  the  serum  treatment. 

It  may  be  readily  understood  with  these  facts  before  us  why  the 
practice  of  protective  vaccinations  of  animals  against  tetanus  should 
have  spread  so  rapidly  amongst  veterinarians.  The  demand  for  anti- 
tetanus  serum  from  the  Pasteur  Institute  of  Paris  for  veterinary  use 
increases  every  year  at  a  great  ratio.  Thus  in  1896  there  were  sent 
out  only  1511  bottles  of  10  c.c.  each,  in  1898  the  number  rose  to 
24,959  bottles,  in  1900  it  exceeded  43,000. 

The  efficacy  of  the  antitetanus  serum  employed  as  a  protective 
agent  can  no  longer  be  questioned,  but  it  must  not  be  forgotten 
that  its  injection  does  not  render  the  treatment  of  the  wounds 
unnecessary.  These  wounds  should  receive  a  rigorous  antiseptic 
cleansing.  All  foreign  bodies  should  be  carefully  extracted ;  other- 
wise the  prolonged  presence  of  tetanus  spores  might  set  up  a  late 
tetanus  after  the  disappearance  of  the  transient  immunity  due  to  the 
serum. 

The  protective  injections  of  antitetanus  serum  into  men  likely  to 
contract  tetanus  are  also  beginning  to  spread.  It  often  happens 
that  bicyclists,  in  falling,  receive  injuries  which  are  contaminated  by 

^  l  Bull.  Acad.  de  med.,  Paris,  1895,  t.  xxxiv,  p.  407 ;  ibid.,  1897,  t.  xxxvm,  p.  109; 
Compt.  rend.  XII  Congr.  Internat.  de  Med.  ct  Moscou,  1897,  t.  vn,  p.  244. 


Protective  vaccinations  495 

horse-dung  or  other  matters  which  may  contain  the  spores  of  tetanus. 
In  these  cases,  as  in  many  other  forms  of  injury,  vaccination  with 
antitetanus  serum  is  indicated.  Thus  it  happens  from  time  to  time 
at  the  Pasteur  Institute  that  injured  persons  come  and  ask  for  a  [518] 
protective  injection  of  serum.  Several  medical  men  and  surgeons  are 
now  accustomed  to  vaccinate  such  of  their  patients  as  have  had  their 
wounds  contaminated  by  earth  or  dung.  All  the  cases  of  this  treat- 
ment which  have  come  to  our  knowledge  have  been  followed  by  very 
good  results. 

XII.  Vaccinations  against  diphtheria.  Antidiphtheria  vaccinations 
have  been  the  subject  of  much  discussion  since  the  discovery  of  the 
antidiphtheria  serum  and  its  introduction  into  routine  practice.  A 
large  number  of  works  were  published  for  and  against  the  application 
of  serum  in  protective  treatment  against  diphtheria,  especially  in  the 
early  years  of  its  use.  Later  the  controversy  has  subsided  somewhat, 
and  at  present  very  few  writers  are  found  who  continue  to  decry 
antidiphtheria  vaccinations. 

The  antidiphtheria  serum  was  discovered  in  1890  by  von  Behring 
working  in  collaboration  with  Kitasato ;  these  observers  demonstrated 
in  laboratory  animals  its  neutralising  action  upon  the  diphtheria  toxin. 
A  little  later  von  Behring  began  to  apply  it  in  the  treatment  of 
diphtheria,  but  the  early  results  were  far  from  satisfactory,  and 
von  Behring  soon  recognised  that  it  was  necessary  to  obtain  much 
more  active  serum.  Along  with  Ehrlich  of  the  Institute  for  Infective 
Diseases  at  Berlin  he  set  to  work  to  study  this  problem.  In  colla- 
boration with  several  investigators,  among  whom  I  may  cite  Wernicke, 
Wassermann,  and  Kossel,  he  succeeded  in  obtaining  very  encouraging 
results  as  regards  the  antitoxic  strength  of  the  serums  and  their 
therapeutic  action  on  children  attacked  by  diphtheria. 

At  this  time,  also,  Roux  in  Paris  began,  assisted  by  Martin  and 
Chaillou,  to  study  the  same  question.  These  observers  prepared 
serums  which  for  that  period  were  very  active  and  made  a  very 
effective  application  of  them  upon  more  than  300  diphtheria  patients. 

From  the  year  1894  the  use  of  serum  began  to  spread  in  all 
countries,  and  it  was  then  that  an  attempt  was  made  to  apply  it  to 
the  protection  of  children  in  good  health,  but  who  had  been  specially 
exposed  to  contagion. 

It  was  necessary  to  have  at  command  large  supplies  of  anti- 
diphtheria  serum;  this  was  prepared  by  injecting  into  horses 


496  Chapter  XV 

repeated  doses  of  the  toxin  manufactured  by  the  diphtheria  bacillus. 
[519]  The  serums  thus  obtained  were  first  tested  as  to  their  protective, 
antitoxic,  and  curative  action  on  guinea-pigs,  animals  very  susceptible 
to  diphtheria.  The  necessity  of  finding  some  means  of  measuring 
the  strength  of  the  serum  soon  arose.  Von  Behring  and  Wernicke 
at  first  standardised  it  on  the  basis  of  the  number  of  grammes  of 
guinea-pig  which  could  be  protected  by  one  gramme  of  serum.  Later, 
von  Behring1  introduced  the  principle  of  the  "  normal  serum/'  that 
is  to  say,  a  serum  of  which  O'l  c.c.,  mixed  with  10  lethal  doses  of 
diphtheria  toxin,  is  capable  of  preventing  every  morbid  symptom  in 
a  guinea-pig  weighing  300  to  400  grammes. 

Ehrlich2  perfected  this  method  in  the  following  way:  to  tubes, 
each  containing  10  lethal  doses  of  a  standard  toxin,  are  added 
different  amounts  of  serum.  These  mixtures  are  brought  to  the 
same  volume  of  4  c.c.  by  the  addition  of  physiological  saline  solution, 
and  each  is  immediately  injected  below  the  skin  of  a  guinea-pig.  If 
0*1  c.c.  of  a  serum  completely  neutralises  the  10  lethal  doses  of  toxin, 
the  serum  retains  its  name  of  normal  serum ;  in  the  case  where  0'05  c.c. 
is  sufficient  to  bring  about  the  same  result  the  serum  is  designated 
double  normal  serum.  When  0*001  c.c.  gives  the  same  results,  a 
hundred  times  normal  serum,  and  so  on.  A  cubic  centimetre  of 
normal  serum  (that  is  to  say  a  dose  capable  of  neutralising  100  lethal 
doses  of  standard  toxin)  constitutes  an  "  immunising  unit "  (Immuni- 
sirungseinheit  (I.E.)  of  Ehrlich).  As  it  was  soon  recognised  that  toxins, 
even  when  kept  under  the  best  conditions,  lose  more  or  less  of  their 
toxic  power,  Ehrlich  had  to  modify  his  method  of  standardising 
serum.  He  now  makes  use  of  a  standard  antidiphtheria  serum,  kept 
in  a  dry  condition,  which  is  much  more  constant  than  are  the  toxins. 
Solutions  of  this  standard  serum  are  prepared  and  compared  with 
the  serum  whose  strength  has  to  be  determined.  Ehrlich  has  given  a 
detailed  description  of  the  method  of  procedure  required  to  obtain 
exact  results. 

At  the  Pasteur  Institute  Ehrlich's  method  has  been  adopted, 
supplemented  however  by  another  test  for  the  estimation  of  the 
strength  of  antidiphtheria  serums,  a  method  allied  to  von  Behring's 
old  method.  Various  doses  of  the  serum  to  be  examined  are  injected 
subcutaneously  into  guinea-pigs,  and  24  hours  later  these  guinea-pigs 

1  Deutsche  med.  Wclmschr.,  Leipzig,  1893,  8.  390. 

8  Ehrlich,  Kosselu.  Wassermann,  Deutsche  med.  Wchnschr.,  Leipzig,  1894,  S.  353; 
Klin.  Jahrb.j  Berlin,  1897,  Bd.  vi. 


Protective  vaccinations  497 

receive  a  quantity  of  a  living  culture  of  diphtheria  bacilli  which  kills  [520] 
control  animals  in  30  hours.  The  protective  power  of  the  serum 
in  relation  to  the  weight  of  the  animal  is  thus  determined.  For 
example,  a  serum  which  is  said  to  be  active  at  1/100,000  has  the 
power,  in  a  quantity  equal  to  l/100,000th  of  the  weight  of  the 
inoculated  guinea-pig,  of  preventing  a  fatal  result.  It  was  thought, 
at  first,  that  the  protective  power,  measured  in  this  way,  would 
be  proportional  to  the  antitoxic  property  determined  according  to 
Ehrlich's  method.  But  as  the  results  given  by  these  two  methods 
were  often  widely  different,  it  was  resolved  at  the  Pasteur  Institute 
to  examine  by  both  methods  all  the  serums  intended  for  use  in 
practice.  This  led  to  the  conclusion  formulated  by  Roux1,  in  his 
report  communicated  to  the  International  Congress  of  Hygiene,  held 
at  Paris  in  1900,  that  a  serum  possessing  a  very  high  protective 
power  (against  the  living  diphtheria  bacillus)  might  be  only  feebly 
antitoxic,  and  vice  versd. 

This  result  is  explained  by  the  fact  that  the  antidiphtheria  serums 
are  very  complex  fluids,  containing  several  superposed  properties 
of  very  variable  strength.  Marx2,  of  the  Frankfort-on-Main  Institute, 
tried  to  shake  Roux's  conclusions,  bringing  forward  his  experiments 
made  on  guinea-pigs  and  rabbits  injected  with  antidiphtheria  serum 
into  the  peritoneal  cavity  and  into  the  veins.  He  wished  in  this  way 
to  avoid  the  introduction  of  the  serum  into  the  subcutaneous  tissues, 
whence  the  absorption  of  the  antitoxin  must  take  place  in  a  very 
irregular  fashion.  In  Marx's  experiments,  thus  carried  out,  the 
protective  power  of  the  serums  was  always  found  to  run  parallel  with 
their  antitoxic  power,  from  which  he  concluded  that  Roux's  view 
was  incorrect.  It  must  not  be  forgotten,  however,  that  this  view 
was  founded  on  experiments  in  which  the  antitoxin  had  been 
injected  into  the  subcutaneous  tissue  before  or  simultaneously  with 
the  toxin  or  the  diphtheria  bacillus.  Under  these  conditions  the 
protective  power  is  often  found  to  be  altogether  disproportionate  to 
the  antitoxic  power.  This  fact  has  been  observed  so  carefully  and  with 
such  exactness  that  it  is  impossible  to  deny  it.  Xow  it  is  undoubted 
that  the  conditions  of  the  experiments  upon  which  Roux  relies 
correspond  much  more  closely  with  those  that  are  realised  in 
vaccination  of  man  against  diphtheria  than  with  the  conditions  met 
with  in  Marx's  experiments.  In  these  vaccinations  antidiphtheria 

1  Compt.  rend.  X  Congr.  internal,  d'hyg.  et  de  demogr.,  Paris,  1900. 

2  Ztschr.  d.  Hyg.,  Leipzig,  1901,  Bd.  xxxvin,  S.  372. 

B.  32 


498  Chapter  XV 

[521J  serum  is  injected  below  the  skin  of  persons  whom  it  is  wished  to 
protect  against  the  action  of  the  diphtheria  bacillus. 

With  the  object  of  bringing  about  a  unification  of  the  methods 
of  estimating  serums  used  in  different  countries  the  International 
Congress  of  Hygiene,  held  at  Madrid  in  1898,  appointed  a  special 
Commission  to  settle  this  problem.  But  when  the  Congress  met 
again  at  Paris  in  1900  this  Commission  had  not  completed  the  task 
allotted  to  it.  The  representatives  of  the  various  methods  had 
exchanged  ideas,  but  in  applying  the  same  method  the  results 
obtained  in  various  places  and  by  various  observers  presented 
differences  too  great  to  allow  of  any  understanding  being  arrived  at. 
It  is  evident  that  we  have  here  a  very  complicated  problem.  The 
serums  are  tested  on  living  animals  in  which  of  course  nothing  like 
the  constancy  of  a  chemical  reaction  can  be  obtained. 

Possibly  the  methods  of  breeding  and  the  races  of  the  same 
animals  in  the  different  countries  may  be  quite  sufficient  to  explain 
the  divergencies  in  the  results  obtained.  Whatever  may  be  the 
.  reason  the  unification  of  serum  estimation  has  not  yet  been  obtained, 
and  it  is  difficult  to  anticipate  that  any  better  result  is  to  be  ar- 
rived at. 

From  all  this  we  may  draw  the  conclusion  that  the  possibility 
of  attaining  a  too  rigorous  precision  in  the  standardisation  of  serum 
has  been  exaggerated.  Our  object  must  be  to  obtain  results  as 
favourable  as  possible  in  the  application  of  the  antidiphtheria  serums, 
and  for  that  purpose  it  is  necessary  to  inject  greater  quantities  than 
those  which  may  be  indicated  by  any  method  of  estimation.  This  rule 
is  applied  as  far  as  is  possible  at  the  Pasteur  Institute. 

As  regards  vaccination  against  diphtheria  of  persons  who  are  in 
good  health  but  are  especially  exposed  to  infection,  the  question 
must  be  accepted  as  settled  in  the  affirmative. 

From  the  commencement  of  our  attempt  to  cure  diphtheria  by 

means  of  a  specific  serum,  the  necessity  was  seen  of  protecting 

children  who  were  in  contact  with  the  sick  persons  against  this 

disease.    Small  quantities  of  serum  were  injected  into  such  children 

for  protective  purposes.    The  first  results  communicated  in  1894  by 

Roux  to  the  Congress  at  Budapest  being  very  encouraging,  an  attempt 

was  made  to  give  the  greatest  possible  extension  to  the  system  of 

vaccination  by  antidiphtheria  serum.  In  the  following  year,  1895,  fairly 

[522]  numerous  statistics  had  been  collected,  and  Torday1  at  Budapest, 

1  Deutsche  med.  Wchnschr.,  Leipzig,  1895,  S.  408. 


Protective  vaccinations  499 

Kurth1  at  Bremen,  and  Rubens2  at  Gelsenkirchen  were  able  to  publish 
a  number  of  favourable  statistics.  Soon  afterwards,  however,  a  fatal 
case  occurred  in  the  family  of  a  well-known  Berlin  doctor,  Langerhans3, 
an  accident  that  started  a  violent  controversy  and  stirred  up  an  active 
campaign  against  serum.  Langerhans's  son,  a  boy  aged  2  years,  in 
good  health,  was  inoculated  with  a  small  dose  (1*2  c.c.  of  this  serum) 
and  succumbed  about  a  quarter  of  an  hour  afterwards  with  symptoms 
of  suffocation.  The  post-mortem  examination  made  by  Strassman4 
showed  the  cause  of  death  to  be  suffocation  in  consequence  of  the 
aspiration  of  food  into  the  respiratory  passages  during  the  act  of 
vomiting.  An  examination  of  the  serum  used  by  Langerhans  did 
not  reveal  any  toxic  action  on  animals  or  any  contamination  by 
micro-organisms.  All  to  no  purpose,  the  serum  was  held  answerable 
for  the  death  of  the  child,  and  an  attempt  was  made  to  demonstrate 
at  almost  any  cost  that  its  use  in  human  practice  was  extremely  dan- 
gerous. Gottstein5  joined  in  chorus  with  the  over-excited  opinion  and 
published  a  denunciation  of  vaccinations  by  antidiphtheria  serum.  He 
collected  from  the  literature  of  both  hemispheres  four  cases,  in  all, 
in  which  death  had  occurred  some  time  after  the  injection  of  this 
serum  into  children  not  suffering  from  diphtheria.  A  perusal  of  the 
description  of  these  cases  is  sufficient  to  convince  one  that  the  death 
could  in  no  sense  be  attributed  to  the  serum,  and  that  it  could  be 
explained  much  more  easily  by  the  fatal  action  of  the  streptococcus, 
the  cause  of  the  non-diphtheritic  affections  of  the  children  that 
died. 

The  ineptitude  of  this  denunciation  must  have  done  much  to  calm 
public  opinion,  and  in  September  of  the  same  year,  1896,  C.  Frankel6, 
in  a  report  presented  to  the  German  Association  of  Public  Hygiene, 
was  able  to  give  a  review  of  the  state  of  the  question  of  vaccination 
against  diphtheria,  summing  up  in  favour  of  the  use  of  the  specific 
serum.  "  Taking  into  consideration  the  data  collected,"  he  remarks, 
41  it  is  scarcely  possible  to  doubt  the  value  of  immunisation  by  serum,  [523] 
so  that  we  may  say  positively  that  we  are  now  treading  a  path  which 
will  lead  us  to  great  and  important  results."  This  very  favourable 

1  Deutsche  med.  Wchnschr.,  Leipzig,  1895,  SS.  426,  443,  464. 

2  Deutsche  med.  Wchnschr.,  Leipzig,  1895,  S.  758. 

3  Berl  klin.  Wchnschr.,  1896,  S.  602. 

4  Berl.  klin.  Wchnschr.,  1896,  S.  516. 

6  Therap.  Monatsh.,  Berlin,  1896,  S.  269. 

6  Deutsche  Vrtljschr.f.  off.  Gsndhtspflg.,  Brnschwg.,  1897,  Bd.  xxix,  Heft  1. 

32—2 


500  Chapter  XV 

opinion  was  due  in  great  measure  to  the  vaccinations  carried  out  in 
the  wards  of  Heubner's  Clinic  at  Berlin1.  At  first,  injection  of  the 
antidiphtheria  serum  as  a  protective  into  patients  who  were  found 
in  the  immediate  vicinity  of  the  children  attacked  with  diphtheria 
(contacts)  was  deemed  to  be  sufficient :  but  in  consequence  of  the 
results  obtained  by  this  method  it  was  decided  (starting  from  January, 
1896)  to  inject  all  children  who  came  into  the  hospital.  During  the 
first  period  there  still  occurred  a  few  cases  of  diphtheria  contracted 
in  hospital,  but  from  the  moment  systematic  and  general  vaccinations 
were  introduced  not  a  single  new  case  occurred. 

The  immune  condition  of  the  vaccinated  children  is  maintained 
for  three  to  four  weeks.  After  this  lapse  of  time  some  of  them 
contracted  diphtheria.  But  it  was  sufficient  to  introduce  revacci- 
nation  at  the  end  of  this  period  to  prevent  the  outbreak  of  any 
further  case  of  diphtheria  in  Heubner's  wards.  Results  quite  as 
favourable  and  as  convincing  were  obtained  in  the  department  for 
children  attacked  by  scarlet  fever. 

The  amount  of  serum  injected  varied,  but  it  was  usually  given  in 
doses  of  1  c.c.  containing  from  200  to  250  I.E.  (immunising  units  of 
Ehrlich).  The  serum  was  always  found  to  be  innocuous  except  in 
certain  cases  where  it  set  up  erythemata  of  greater  or  less  extension. 
In  460  injections  20  cases  of  these  exanthemata  were  produced,  that 
is  to  say  4*34  %•  The  frequency  of  these  complications  was  not  pro- 
portional to  the  amount  of  serum  injected.  According  to  the  figures 
communicated  by  Lbhr  the  largest  doses  of  the  serum  employed 
did  not  produce  exanthemata  more  frequently  than  did  the  smaller 
quantities.  Thus  117  injections  of  1  c.c.  only  were  followed  in  five 
cases  by  these  erythemata,  which  corresponds  to  4*27  per  cent.  The 
hope  of  diminishing  the  frequency  of  the  exanthemata  by  diminishing 
the  amount  of  serum  injected  was  therefore  not  realised.  This 
fact  lends  support  to  the  conclusion  above  formulated  as  to  the 
exaggeration  of  the  importance  of  the  measurement  of  serum.  If  it 
could  be  established  that  small  quantities  of  serum  rich  in  antitoxin 
caused  cutaneous  eruptions  less  frequently  than  did  stronger  doses 
[524]  there  would  certainly  be  a  great  advantage  in  using  serums  containing 
a  very  large  number  of  immunising  units  for  vaccination.  Perhaps 
serums  having  a  great  antimicrobial  power  but  of  comparatively 
low  antitoxic  potency  might  even  render  great  service  in  protective 

1  See  the  report  by  Lohr  in  Jahrb.f.  Kinderh.,  Leipzig,  1896,  Bd.  XLIII,  S.  67. 


Protective  vaccinations  501 

treatment.     Future  researches  undertaken  in  this  direction  alone  can 
give  us  information  on  this  subject. 

In  1896  the  vaccinations  in  Heubner's  wards  were  discontinued, 
but  the  reappearance  of  diphtheria  in  18971  rendered  their  recom- 
mencement necessary.  500  children  were  vaccinated  each  with  200 
immunising  units.  Following  this  no  case  of  diphtheria  broke  out. 
The  eruptions  were  rare  and  slight. 

The  increasing  extension  of  the  use  of  antidiphtheria  serum  for  the 
cure  of  the  disease  after  it  has  broken  out  has  led  to  a  greater  de- 
velopment in  its  use  as  a  preventive  measure.  Thus,  in  the  countries 
where  diphtheria  is  endemic,  vaccinations  by  serum  are  now  practised 
very  extensively.  In  Russia,  which  is  one  of  the  great  hotbeds  of  this 
disease,  vaccinations  by  antidiphtheria  serum  are  frequently  practised. 

At  the  Congress  of  Russian  doctors  at  Kasan  in  1896,  Vissotsky 
communicated  the  result  of  2,185  vaccinations  which  gave  a  morbidity 
of  1*3%,  a  morbidity  that  must  be  regarded  as  very  low  indeed. 
A  well-known  Russian  physician  for  children's  diseases,  Rauchfuss2, 
who  cites  these  figures,  has  collected  several  other  facts  concerning 
the  prophylactic  injections  of  antidiphtheria  serum  followed  by  good 
results.  In  the  government  of  Woronetz,  according  to  the  state- 
ments of  Ouspensky3,  out  of  738  vaccinated  persons  diphtheria 
occurred  in  2'2  per  cent.,  which  again  may  be  considered  a  favourable 
result,  especially  if  we  take  into  account  the  great  extension  of 
diphtheria  in  this  country.  In  Podolia,  out  of  537  children  vacci- 
nated in  1895,  only  four  cases  of  diphtheria  occurred,  a  morbidity 
of  0*74  %.  In  the  government  of  Kherson,  one  of  the  great  centres 
of  diphtheria  in  southern  Russia,  the  results  appear  to  be  less 
favourable :  out  of  543  children  which  received  a  protective  inocu- 
lation, 21  contracted  the  disease  (or  4*6  per  cent.),  of  which  five  died. 
If  we  study  these  statistics  more  closely4  it  will  be  seen  that  these 
results  are  far  from  being  unfavourable.  The  protective  inoculations  [525] 
were  made  only  once  and  with  somewhat  small  doses,  nevertheless 
many  of  the  cases  of  diphtheria  broke  out  only  at  a  late  period,  some- 
times more  than  nine  months  after  the  injections  had  been  made. 
Now,  it  is  proved  that  these  injections,  although  very  efficacious, 

1  See  Slawyk.,  Deutsche  med.  Wchnschr.,  Leipzig,  1898,  S.  35. 

2  "Les  progres  dans  Tapplication  du  serum  antidiphtherique,"  St  Petersbourg, 
1898,  p.  105    in  Russian). 

3  Vrach,  St  Petersbourg,  1900,  p.  1178  (in  Russian). 

4  Chron.  med.  d.  gouvern.  de  Kherson,  1896,  No.  5,  p.  160  (in  Russian). 


502  Chapter  XV 

produce  their  action  for  a  very  short  time  only,  for  a  few  weeks  at 
most.  Of  the  five  fatal  cases,  four  did  not  occur  until  2,  4J,  6,  and 
9J  months  respectively  after  the  protective  inoculation.  It  is  im- 
possible to  look  upon  these  statistics  as  affording  proof  of  the 
inefficacy  of  the  serum.  The  fifth  case  is  the  only  one  that  occurred 
within  a  short  time  (15  days)  of  the  injection,  and  in  this  instance 
only  150  immunising  units  had  been  injected. 

A  detailed  study  of  the  other  examples  of  antidiphtheria  inocu- 
lations in  the  government  of  Kherson  leaves  a  very  favourable  im- 
pression. Out  of  90  children  inoculated  by  Wecker1  in  the  district 
of  Elisabetgrad  not  a  single  one  contracted  diphtheria,  which  is  the 
more  remarkable  as  at  the  time  of  the  inoculations  there  existed  in 
the  same  families  14  cases  of  diphtheria;  the  chances  of  contamination 
were  thus  great. 

Recently,  on  the  occasion  of  the  outbreak  of  a  great  epidemic  in 
Paris,  the  question  of  vaccinations  by  serum  was  again  raised  and 
earnestly  discussed  at  the  Paris  Hospitals  Medical  Society  and  at  the 
Society  for  the  Study  of  Children's  Diseases.  Voisin  and  Guinon2 
communicated  the  history  of  an  epidemic  amongst  the  staff  at  the 
Salpetriere  Hospital  in  the  wards  of  idiot  children,  "against  which 
protective  serum  treatment  was  remarkably  effective  and  absolutely 
innocuous."  The  serum  was  injected,  in  the  case  of  children  more 
than  10  years  of  age,  in  10  c.c.  doses,  and  into  the  rest  in  6  c.c.  doses. 
This  measure  brought  about  first  an  abatement  and  then  cessation 
of  the  epidemic.  The  immunity  after  a  single  injection  lasted  from 
two  to  three  weeks,  and  the  few  cases  of  diphtheria  which  broke  out 
amongst  the  infected  children  were  distinguished  by  their  great 
mildness.  Erythemata  and  other  post-injection  complications  were 
insignificant,  so  that  the  protective  use  of  the  serum  was  fully 
justified.  Only  a  small  minority  of  the  medical  men  who  took  part 
in  the  discussion  spoke  against  the  antidiphtheria  vaccinations ; 
once,  indeed,  a  reference  was  made  to  the  case  of  Langerhans's  child, 
although  its  death  was  certainly  not  due  to  the  serum.  It  is  true 
that  in  families  where  it  is  possible  to  keep  the  children  under 
careful  observation  and  to  intervene  at  the  appearance  of  the  first 
[526]  symptoms  of  diphtheria,  the  preventive  injections  may  be  dispensed 
with,  but  in  practice  these  favourable  conditions  are  rarely  realised, 

1  Chron.  mtd.  d.  gouvern.  de  Kherson,  1896,  No.  19,  p.  743. 
*  Bull,  et  mem.  Soc.  med.  des  Hop.  de  Paris,  1901,  p.  585. 


Protective  vaccinations  503 

and  the  prophylactic  serum  treatment  is  then  of  great  service  in 
preventing  the  outbreak  of  the  disease. 

Xetter1  communicated  to  the  Society  of  Pediatrics  a  summary 
of  32,484  observations  on  the  prophylactic  injection  of  antidiphtheria 
serum.  Of  this  number  192  cases  were  noted  in  which  the  diphtheria 
broke  out  in  spite  of  the  injections,  corresponding  to  0*6  per  cent 
of  those  treated.  These  figures,  however,  included  all  cases  of  the 
disease  which  occurred  up  to  thirty  days  after  the  injection.  Now, 
the  immunity  is  often  less  durable  than  this,  and  it  may  disappear 
more  or  less  completely  twenty  days  and  sometimes  even  fifteen  days 
after  vaccination. 

Netter  himself  made  great  use  of  antidiphtheria  vaccination. 
It  was  his  custom  to  propose  to  the  parents  either  a  protective 
inoculation  at  once  or  a  systematic  precautionary  bacteriological 
examination  of  the  throats  of  the  children  not  yet  attacked.  He 
regards  the  first  method  as  preferable.  According  to  the  latest 
statistics  which  he  was  kind  enough  to  communicate  to  me,  of 
152  children  (in  50  families),  91  of  whom  received  protective  inocu- 
lations, not  one  contracted  diphtheria :  whilst  in  239  other  families 
where  the  children  had  not  been  inoculated  there  were  52  cases  of 
diphtheria,  with  10  deaths.  Many  practitioners  in  Paris  have  now 
pronounced  themselves  in  favour  of  protective  injections  of  the  serum, 
and  the  Society  of  Pediatrics,  at  its  meeting  on  llth  June,  1901, 
concluded  the  discussion  of  this  question  by  proposing  the  following 
resolution :  "  The  Society  of  Pediatrics,  affirming  that  protective 
inoculations  present  no  serious  danger  and  confer  a  very  considerable 
amount  of  immunity  for  some  weeks,  recommend  their  use  when 
children  are  gathered  together  in  numbers,  and  in  families  where 
a  scientific  supervision  cannot  be  maintained/' 

The  large  amount  of  evidence  collected  on  this  question  leaves  no 
doubt  as  to  the  real  efficacy  of  vaccinations  by  antidiphtheria  serum. 

The  summary  of  the  results  obtained  by  vaccination  in  the 
12  diseases  of  man  and  of  animals  I  have  just  placed  before  my 
readers  cannot  pretend  to  serve  as  a  detailed  guide  to  prophylactic 
practice.  My  object  has  been  merely  to  concentrate  into  one  chapter  [527] 
the  principal  data  upon  which  this  very  important  question  rests,  to 
bear  witness  to  the  progress  which  has  already  been  realised,  and  at 
the  same  time  to  show  that  the  scientific  study  of  immunity  is  in 

1  Bull.  Soc.  d.  Pediatr.  de  Paris,  1901,  mai  et  juin. 


504  Chapter  XV 

very  intimate  relation  with  its  practical  application.  It  is  evideni 
that  the  road  is  far  from  traversed  to  its  terminus,  for  there  are 
many  infective  diseases  in  which  vaccinations  cannot  be  employed, 
but  it  is  none  the  less  certain  that  the  path  which  has  led  to  so 
many  important  and  useful  results  should  still  be  followed  in  studying 
problems  which  up  to  the  present  we  have  been  unable  to  solve. 


CHAPTER  XVI  [528] 

HISTORICAL  SKETCH  OF  OUR  KNOWLEDGE 
ON  IMMUNITY 

Methods  used  by  savage  races  for  vaccination  against  snake  venom  and  against 
bovine  pleuropneumonia. — Variolisation  and  vaccination  against  small-pox. — 
Discovery  of  the  attenuation  of  viruses  and  of  vaccinations  with  attenuated 
micro-organisms. — Theory  of  the  exhaustion  of  the  medium  as  a  cause  of 
acquired  immunity. — Theory  of  substances  which  prevent  the  multiplication 
of  micro-organisms  in  the  refractory  body.— Local  theory  of  immuuity.— Theory 
of  the  adaptation  of  the  cells  of  the  immunised  organism. 

Observations  on  the  presence  of  micro-organisms  in  the  white  corpuscles. — History 
of  phagocytosis  and  of  the  theory  of  phagocytes.— Numerous  attacks  upon  this 
theory. — Theory  of  the  bactericidal  property  of  the  body  fluids. — Theory  of  the 
antitoxic  power  of  the  body  fluids. — Extracellular  destruction  of  micro- 
organisms.— Analogy  between  bacteriolysis  and  haemolysis. — Theory  of  side- 
chains. 

Progress  of  the  theory  of  phagocytes. — Attempts  to  reconcile  it  with  the  humoral 
theory. — Present  phase  of  the  question  of  immunity. 

As  protection  against  disease  is  one  of  the  most  important  amongst 
those  questions  which  are  engrossing  the  attention  of  humanity,  it  is 
natural  that  very  great  attention  should  have  been  devoted  to  it 
from  the  most  remote  times.  We  see  primitive  races,  the  ordinary 
layman,  medical  men,  legislators  and  even  the  most  subtle  thinkers 
devoting  their  energies  to  the  solution  of  the  problem  of  immunity 
against  poisoning  and  against  infections.  Historical  science  will 
never  reveal  to  us  the  earliest  sources  of  our  knowledge  on  this 
question,  so  remote  are  their  origins.  The  wide  distribution  of 
several  methods  for  protecting  man  and  cattle  against  certain  diseases 
clearly  proves  that  the  origin  of  this  practice  dates  from  a  very  early 
period. 

The  frequency  of  venomous  snakes  in  many  countries  has  inspired 
a  dread  of  these  reptiles,  and  this  must  have  led  to  the  search  for 


506  Chapter  XVI 

some  method  of  fighting  against  the  poisoning  after  the  patient 
had  been  bitten.  Thus,  we  find  that  many  primitive  races  make  use 
of  various  methods  of  immunising  the  body  against  the  action  of 
[529]  venom.  The  Portuguese  colonel,  Serpa  Pinto1,  in  a  letter  addressed  to 
d'Abbadie,  describes  the  method  by  which  he  was  vaccinated  by  the 
Vatuas,  natives  of  the  east  coast  of  Africa.  These  savages  extract 
the  poison  of  snakes  and  prepare  from  it,  by  the  addition  of  vegetable 
substances,  a  very  brown  glutinous  paste  which  they  introduce  into 
incisions  made  in  the  skin.  This  operation  is  very  painful  and  is 
followed  by  a  swelling  which  lasts  for  a  whole  week.  The  Vatuas 
assert  that  this  method  confers  a  sure  immunity  against  the  venom. 
Serpa  Pinto  was  never  bitten  by  a  snake,  but,  a  short  time  after 
he  had  been  vaccinated,  he  was  stung,  in  the  Seychelles  Islands,  by 
a  scorpion  without  experiencing  any  ill  effects.  This  experience 
confirms  the  assertion  of  the  Vatuas,  because  it  has  been  shown  that 
the  vaccine  against  snake  venom  is  also  efficacious  against  the  bite 
of  scorpions.  The  fact  that  after  being  stung  by  another  scorpion 
ten  years  later  Serpa  Pinto  was  so  ill  that  for  eight  days  he  believed 
that  he  was  going  to  die  or  at  least  to  lose  an  arm,  shows  that  he  did 
not  enjoy  natural  immunity,  and  the  innocuousness  of  the  previous 
bite  must  therefore  be  attributed  to  a  vaccination  the  effect  of  which 
had  disappeared  at  the  end  of  ten  years. 

Another  vaccinal  method  used  by  primitive  races  is  that  against  the 
pleuropneumonia  of  the  Bovidae.  De  Rochebrune2  points  out  that 
the  Moors  and  the  Pouls  of  Senegambia  have  "a  custom  whose 
origin  is  lost  in  the  obscurity  of  antiquity"  which  consists  in  the 
inoculation  into  their  herds  of  cattle  of  the  virus  of  the  epizootic 
pleuropneumonia.  "The  point  of  a  knife  of  primitive  form,  or  of 
a  dagger,  is  plunged  into  the  lung  of  an  animal  that  has  died  from 
the  disease  and  an  incision,  sufficient  to  allow  the  virus  to  penetrate 
below  the  skin  of  the  healthy  animal,  is  made  into  the  supranasal 
region.  Experience  has  demonstrated  the  success  of  this  protective 
operation." 

In  Europe,  the  vaccinations  of  cattle  with  the  virus  of  pleuro- 
pneumouia  have  certainly  been  known  for  more  than  a  century,  for, 
in  a  pamphlet  published  at  Berne  in  1773s,  mention  is  made  of  the 
"inoculation"  of  Bovidae  as  a  means  of  preventing  the  disease  in 

1  Compt.  rend.  Acad.  d.  sc.t  Paris,  1896,  t.  oxxn,  p.  441. 

2  Compt.  rend.  Acad.  d.  sc.,  Paris,  1885,  t.  o,  p.  659. 

8  This  pamphlet  has  been  reprinted  in  the  Rec.  de  med.  vet.,  Paris,  1886,  p.  624. 


Historical  sketch  on  Immunity  507 

England    and    in    Holland,   a    disease    against  which   it   has  been 
recognised  that  remedies  are  powerless. 

The  inoculation  of  the  variolous  virus  into  the  healthy  human  [530] 
subject,  which  comes  into  the  same  category  as  the  inoculation  of  the 
pleuropneumonic  virus  into  healthy  bovine  animals,  is  also  a  widely 
extended  and  very  ancient  method.  The  Chinese1  assert  that  they 
have  known  from  the  commencement  of  the  llth  century  the  method 
of  immunising  against  small-pox.  Amongst  them,  as  amongst  the 
Siamese,  the  matter  from  the  variolous  scab  is  introduced  into  the 
nostrils.  In  Persia  variolisation  is  practised  by  surgeons  and  by  the 
staffs  of  bathing  establishments,  who  introduce  the  powdered  scabs 
into  scratches  in  the  skin.  The  Ashantis  inoculate  the  variolous  virus 
into  seven  places  on  the  amis  and  legs.  According  to  the  account 
of  Timoni,  a  Greek  physician  practising  in  Constantinople  in  the  first 
half  of  the  18th  century,  the  Circassians  and  Georgians,  intent  upon 
preserving  the  beauty  of  their  daughters,  make  punctures  at  various 
points  in  the  skin,  with  needles  charged  with  variolous  virus.  Every- 
body is  acquainted  with  the  fact  that  it  was  from  Constantinople 
that  Lady  Mary  Wortley  Montague  at  the  same  period  (1721),  im- 
ported into  Europe  "the  Greek  method,"  which  consisted  in  the 
inoculation  of  the  contents  of  small-pox  pustules  with  the  object  of 
producing  a  benign  small-pox  and  of  protecting  the  vaccinated 
person  from  severe  and  dangerous  small-pox.  This  practice  was 
widespread  in  Europe  during  the  second  half  of  the  18th  century, 
but  as  it  was  not  unattended  by  serious  drawbacks  an  attempt  was 
made  to  avoid  them  by  the  employment  of  all  kinds  of  medica- 
ments. As  these,  however,  were  found  to  be  entirely  ineffective,  the 
need  was  felt  of  replacing  variolisation  by  some  more  benign  method. 

It  is  asserted2  that  in  Baluchistan  the  custom  of  having  cows 
suffering  from  cow-pox  milked  by  children  who  had  wounds  on  their 
hands  has  been  widespread  from  time  immemorial.  This  practice 
conferred  upon  these  children  an  immunity  against  small-pox.  It 
cannot  be  denied  that  the  idea  of  being  able  to  vaccinate  with  cow- 
pox  was  common  knowledge  amongst  breeders  and  dairymen  in  several 
countries  in  Europe,  especially  in  England,  France,  and  Germany. 
It  is  stated  that  Edward  Jenner  learnt  from  the  country  people  of 
his  native  county  of  Gloucestershire  that  contact  with  cow-pox 

1  Barthels,  "Die  Medicin  der  Naturvolker,"  Leipzig,  1893,  S.  128;  Pagel,  "Ein- 
fuhrung  in  die  Geschichte  der  Medicin,"  Berlin,  1898,  S.  313. 

2  Baser,  "Lehrbuch  der  Geschichte  der  Medicin,"  3te  Aufl.,  Jena,  1881,  Bd.  n. 
S.  1075. 


508  Chapter  XVI 

protected  against  small-pox.  Being  a  man  of  great  understanding  and 
culture,  he  set  himself  to  verify  this  opinion  experimentally.  Having 
[531]  demonstrated  by  a  great  number  of  experiments  that  the  inoculation  of 
variolous  virus  into  persons  vaccinated  by  cow-pox  had  no  ill  result, 
he  became  the  great  propagandist  of  the  new  method.  He  worked 
at  this  subject  for  20  years  but  only  decided  to  publish  his  results  (in 
1798)  after  he  had  completely  satisfied  himself  of  the  great  utility 
of  vaccination  with  the  virus  of  cow-pox.  At  first  Jenner's  discovery 
met  with  great  opposition,  but  his  method  was  soon  verified  in 
France  and  several  other  countries  and  it  was  not  long  before  it  was 
generally  practised. 

When  Pasteur  set  himself  to  study  the  infective  diseases  in 
their  relation  to  micro-organisms  the  idea  of  profiting  by  the 
discovery  of  these  pathogenic  organisms  and  of  drawing  from  them 
a  weapon  against  infections  soon  arose  in  his  mind.  He  studied 
Jenner's  work  in  order  to  extract  from  it  any  indications  capable 
of  putting  him  into  the  right  path.  He  induced  his  collaborators 
to  carry  out  several  series  of  experiments  with  the  object  of  immu- 
nising the  animal  organism  against  infective  micro-organisms.  During 
this  laborious  and  original  work  chance1  helped  in  the  accom- 
plishment of  his  task.  When,  at  the  conclusion  of  the  holidays  in 
the  autumn  of  1879,  Pasteur  and  his  collaborators  Chamberland  and 
Roux  wished  to  resume  their  experiments  on  fowl  cholera,  they 
found  to  their  great  surprise  that  the  micro-organisms  of  this  disease, 
usually  so  fatal,  had  become  innocuous.  Fowls,  that  received  doses 
of  cultures  much  more  than  sufficient  to  cause  death,  did  not 
experience  any  ill  effect.  Prepared  by  his  previous  knowledge  and 
by  the  continual  direction  of  his  thoughts  to  the  prevention 
of  contagious  diseases,  Pasteur  divined  at  once  the  great 
bearing  of  this  check  in  his  inoculations  with  old  cultures,  and 
immediately  began  to  make  precise  experiments  as  to  the 
vaccinating  power  of  these  micro-organisms  which  had  become 
innocuous.  These  researches  led  him  to  the  discovery  of  two  great 
principles:  that  of  the  attenuation  of  viruses,  and  that  of  the 
vaccinating  property  of  attenuated  micro-organisms.  Various  me- 
moirs by  Pasteur2  established  these  laws  in  a  very  exact  manner ; 
moreover  he  gave  all  the  information  necessary  to  allow  of  the 

1  See  Vallery-Radot,  "La  Vie  de  Pasteur,"  Paris,  1900,  p.  427. 

2  Compt.  rend.  Acad.  d.  sc.,  Paris,  1880,  t.  xc,  pp.  939,  952,  1030;  t.  xcr,  pp.  571, 
673. 


Historical  sketch  on  Immunity  509 

principal  results  being  controlled  and  verified.  In  France,  this  great 
discovery  was  at  once  accepted  by  various  investigators,  though 
others  found  occasion  to  manifest  their  scepticism.  Abroad  this  [532] 
discovery  met  with  very  lively  opposition  and  this  from  the  highest 
authorities,  who  would  not  recognise  the  possibility  either  of  attenuat- 
ing the  virus  or  of  conferring  immunity  upon  animals.  The  anthrax 
bacillus  can  be  grown  for  a  very  long  time  on  culture  media,  the  potato, 
for  example,  without  losing  its  pathogenic  power  in  the  slightest 
degree.  Therefore,  it  was  said,  this  attenuation  of  virus  can  have  no 
actual  existence.  White  rats  that  have  resisted  one  or  more  inocu- 
lations of  the  anthrax  bacillus  may  die  from  a  later  inoculation  of  the 
same  micro-organism.  Therefore  there  is  no  acquired  immunity,  etc. 
The  principles  laid  down  by  Pasteur  are  from  every  point  of  view  of 
such  prime  importance,  that  very  numerous  experiments  were  carried 
out  at  once  for  the  purpose  of  verifying  their  exactness  and  the 
contest  was  not  a  long  one.  In  the  course  of  a  few  years  it  was 
universally  recognised  that  the  attenuation  of  viruses,  and  also  the 
vaccination  by  attenuated  micro-organisms,  were  realities  which 
henceforth  cannot  be  denied  and  which  must  pass  into  the  domain  of 
truths  definitely  acquired.  An  attempt  was  then  made  to  extend  these 
fresh  victories  to  the  other  infective  diseases.  Pasteur,  Chamber-land, 
and  Roux  applied  themselves  to  devising  a  method  of  vaccinating  ani- 
mals against  anthrax  and  against  rabic  virus ;  Pasteur  and  Thuillier 
extended  their  researches  on  this  subject  to  swine  erysipelas. 
From  several  other  quarters  the  search  for  vaccines  was  instituted. 
Toussaint  made  various  attempts,  at  times  croAvned  with  success,  to 
immunise  animals  against  anthrax  by  means  of  heated  anthrax  blood. 
Arloing,  Cornevin,  and  Thomas  succeeded  in  vaccinating  the  Bovidae 
against  symptomatic  anthrax.  Loeffler  was  the  first  in  Germany  to 
demonstrate  that  rabbits  which  had  recovered  from  the  disease  set 
up  by  the  bacillus  of  mouse  septicaemia  acquired  an  immunity 
against  the  attacks  of  this  organism.  It  is  not  necessary  to  cite 
further  examples,  so  numerous  have  they  become  and  so  unanimously 
confirmatory. 

After  the  first  steps  had  been  taken  along  this  new  path  Pasteur 
and  his  collaborators  began  to  apply  the  knowledge  they  had  gained 
to  the  preparation  of  vaccines  capable  of  giving  practical  results. 
The  two  anti-anthrax  vaccines  and  the  two  vaccines  against  swine 
erysipelas  were  the  fruit  of  these  attempts.  Here,  again,  numerous 
objections  were  raised  against  these  discoveries.  Sheep  which  had 


510  Chapter  XVI 

[533]  received  enormous  quantities  of  the  bacillus  may  die  from  anthrax  in 
spite  of  the  two  Pasteurian  vaccines  and  from  that  it  was  wished  to 
conclude  that  these  vaccines  should  not  be  employed  in  practice  to 
protect  sheep  against  the  anthrax  fever.  The  results  of  experiments 
made  on  a  large  scale  in  various  parts  of  the  globe  have  demonstrated 
the  inadequacy  of  these  objections  and  these  questions  are  now 
regarded  as  definitely  settled. 

So  large  a  number  of  investigations,  in  response  to  the  most  urgent 
and  immediate  needs,  was  not  favourable  to  minute  researches  on  the 
mechanism  of  this  immunity  which  had  been  revealed  in  so  marvellous 
a  fashion.  In  spite  of  this,  Pasteur  applied  himself  to  the  solution 
of  this  problem  so  far  as  this  was  possible  under  the  conditions  in 
which  he  carried  on  his  investigations.  He  thought  that  acquired 
immunity  was  the  result  of  the  impossibility  of  the  growth  of  a 
pathogenic  micro-organism  in  a  medium  in  which  ifc  had  previously 
been  cultivated.  When  the  micro-organism  of  fowl  cholera  sets  up  in 
certain  individuals  a  disease  which  though  grave  is  not  fatal,  or 
when  the  attenuated  micro-organism  produces  a  simple,  transient 
discomfort,  it  lives  in  both  cases  in  the  fluids  and  tissues  of  the 
animal.  This  existence  is  possible  in  consequence  of  the  absorption 
of  certain  nutrient  substances.  Once  these  substances  are  consumed 
they  are  not  easily  renewed,  and  in  consequence  the  vaccinated 
organism  becomes  incapable  of  nourishing  the  special  micro-organism 
a  second  or  a  third  time.  To  support  this  brilliant  hypothesis  by 
precise  facts  Pasteur  made  experiments  on  the  conditions  met  with 
in  the  development  of  the  micro-organism  of  fowl  cholera  in  vitro. 
He  filtered  a  broth  culture  of  this  micro-organism  after  it  had  grown 
luxuriantly  for  several  days,  and  into  the  fluid,  which  had  now 
become  clear  and  transparent,  he  sowed  afresh  the  same  micro- 
organism. No  growth  took  place  and  the  fluid  remained  quite  clear. 
This  absence  of  development  might  be  explained  either  by  the 
presence  in  the  fluid  of  some  excremental  substance  thrown  off 
during  the  first  culture  or  by  the  absence  of  some  substance  indis- 
pensable for  the  nutrition  of  the  micro-organism.  Pasteur  excluded 
the  first  hypothesis  by  an  experiment  which  demonstrated  that  it 
is  sufficient  to  add  to  the  filtered  fluid  a  small  quantity  of  fresh 
nutritive  substances  to  enable  the  micro-organism  again  to  develop 
abundantly.  It  is  therefore  to  the  absence  of  some  element  essential 
to  the  existence  of  the  micro-organism  that  we  must  attribute  the 
immunity  enjoyed  by  animals  which  have  been  vaccinated  or  which 


Historical  sketch  on  Immunity  511 

have  undergone  spontaneous  cure.  This  is  how  Pasteur1  expressed 
himself  on  this  point :  "  the  muscle  which  has  been  much  affected  [534] 
has,  even  after  healing  and  repair,  become  in  some  way  incapable 
of  supporting  the  growth  of  the  micro-organism,  as  if  the  latter,  by 
a  previous  culture,  had  eliminated  from  the  muscle  some  principle 
that  life  does  not  bring  back  and  whose  absence  prevents  the 
development  of  the  small  organism.  There  is  no  doubt  that  this 
explanation,  to  which  the  plainest  facts  at  the  moment  lead  us,  will 
become  general  and  applicable  to  all  the  virulent  diseases." 

This  explanation  appeared  to  be  a  reasonable  one  to  several 
observers,  amongst  whom  I  may  cite  Chauveau2,  the  distinguished 
author  of  important  works  on  viruses.  "In  all  probability  this 
seductive  theory,"  says  Chauveau,  "based  on  one  of  the  most  inter- 
esting of  those  clear  and  decisive  experiments  for  which  Pasteur  is 
famous,  applies  to  the  majority  of  cases  of  immunity  acquired  by 
protective  inoculation."  But  Chauveau  thinks  that  it  does  not 
explain  natural  immunity,  especially  that  of  the  Algerian  sheep, 
against  anthrax,  an  example  that  he  had  studied  on  several  occasions. 
When  he  inoculated  into  these  animals  large  quantities  of  anthrax 
bacilli,  not  going  beyond  certain  limits,  the  sheep  resisted  perfectly ; 
but  injections  of  enormous  doses  were  nearly  always  capable  of 
overcoming  this  natural  immunity  of  the  Algerian  sheep  and  of 
inducing  in  them  a  fatal  anthrax.  Chauveau  thinks  that  this  fact 
is  best  explained  by  the  presence  of  an  inhibitory  substance  in  the 
blood  plasma,  whose  action  becomes  exhausted  when  distributed 
over  a  very  large  number  of  bacilli.  This  opinion  was  not,  however, 
shared  by  Pasteur3,  who  raises  the  objection  that  natural  immunity 
can  really  be  produced  and  maintained  without  the  presence  of  this 
inhibitory  substance  from  the  fact  that  fowls,  which  exhibit  such 
marked  resistance  against  anthrax,  readily  contract  the  disease  when 
the  temperature  of  their  bodies  is  lowered.  Under  these  conditions 
it  is  unimaginable  that  an  inhibitory  substance  has  disappeared  under 
the  influence  of  cold. 

The  controversy  existent  from  the  birth  of  theories  on  immunity 
shows  us  that  from  the  very  commencement  the  problem  was  found 
to  be  a  very  complex  one,  and  that  to  attack  it  in  a  satisfactory  way 
we  must  as  far  as  possible  multiply  and  deepen  our  study  of  the 
phenomena  which  accompany  the  resistance  of  the  animal  against 

1  Compt.  rend.  Acad.  d.  sc.,  Paris,  1880,  t.  xc,  p.  247. 

2  Compt.  rend.  Acad.  d.  sc.,  Paris,  1880,  t.  xc,  p.  1526. 

3  Compt.  rend.  Acad.  d.  sc.,  Paris,  1880,  t  xci,  p.  536. 


512  Chapter  XVI 

pathogenic  micro-organisms.  Thus,  Chauveau1  was  not  long  before 
[535]  he  undertook  experiments  having  for  their  object  the  determination 
of  the  fate  of  anthrax  bacilli  when  injected  into  the  blood  vessels  of 
Algerian  sheep.  He  found  that  these  organisms  disappeared  from 
the  blood  at  the  end  of  a  few  hours,  but  they  were  then  to  be  found 
accumulated  in  the  lung,  spleen,  and  certain  other  viscera.  In  these 
positions  the  bacilli  become  incapable  of  reproducing  themselves  and 
in  refractory  individuals  soon  disappear,  being  opposed  by  the  inhibi- 
tory substances  of  the  blood  plasma. 

The  two  theories  just  sketched  have  this  point  in  common,  that 
they  both  attribute  the  natural  or  acquired  immunity  to  humoral 
and  purely  passive  properties.  According  to  one  theory  it  is  the 
impoverishment  of  the  fluids  of  the  animals  which  prevents  the 
development  of  the  pathogenic  organism,  whilst  according  to  the 
other  it  is  the  presence  of  some  bacterial  poison  which  brings  about 
the  same  result.  To  give  experimental  support  to  his  theory  Pasteur 
brought  forward  his  attempts  at  sowing  micro-organisms  in  culture 
media  exhausted  by  a  previous  development  of  the  same  organism, 
eliminating,  so  to  say,  the  active  influence  of  the  animal  organism. 
It  is  true  that,  in  order  to  explain  natural  immunity,  it  was  necessary 
to  ascribe  a  role  to  the  " constitution "  and  to  the  "vital  resistance," 
interpreting  this,  as  Naegeli  had  already  done,  in  the  sense  of  a 
competition  for  the  oxygen  and  the  nutritive  substances  between  the 
parasites  and  the  cells  of  the  body. 

Adopting  this  point  of  view,  Hans  Buchner2,  a  pupil  of  Naegeli, 
attempted  to  gain  a  more  precise  idea  of  the  conditions  under  which 
acquired  immunity  against  infective  diseases  is  set  up.  He  developed 
his  theory  in  various  publications ;  this  theory  consists,  briefly,  in  the 
property  of  the  animal  organism  to  reinforce  the  local  resistance  of  the 
organs  by  means  of  an  inflammatory  reaction.  The  starting-point  of 
this  local  theory  is  the  thesis  that  each  pathogenic  micro-organism 
can  only  manifest  its  pathogenic  action  when  it  enters  the  particular 
organ  in  which  it  is  capable  of  living  and  maintaining  itself.  Thus, 
the  pneumonococcus  can  live  in  the  lungs  only,  the  cholera  vibrio  in 
the  intestines  only,  and  so  on.  Every  time  that  a  pathogenic  micro- 
organism becomes  localised  in  its  special  organ,  an  inflammatory 
action  is  set  up  which  results  in  the  reinforcement  of  the  living 

1  Compt.  rend.  Acad.  d.  sc.,  Paris,  1880,  t  xci,  p.  680. 

"Die  Naegeli'sche  Theorie  d.  Infectionskrankheiten,"  Leipzig,  1877;  "Eine  neue 
Theorie  iiber  Erziel.  v.  Immunitat,"  Miinchen,  1883. 


Historical  sketch  on  Immunity  513 

elements  of  the  organ  in  question.  Inflammation,  therefore,  is 
regarded  by  Buchner  as  a  salutary  reaction,  which  acts,  not  directly  [536] 
on  the  exciting  morbific  cause,  but  through  the  mediation  of  the 
specific  cells  of  the  organs.  This  theory  of  immunity  led  Buchner  to 
propose  arsenical  treatment  as  a  remedy  against  microbial  disease, 
because  arsenic  is,  of  all  drugs,  the  one  capable  of  setting  up  the 
greatest  inflammatory  reaction. 

Another  German  observer,  Grawitz1,  proposes  a  theory  of  acquired 
immunity,  according  to  which  a  first  attack  of  an  infective  disease 
sets  up  "the  adaptation  of  the  cells  to  the  power  of  energetic 
assimilation  of  the  fungi/'  This  reinforced  adaptation  is  transmitted 
to  the  descendants  of  the  cells  which  have  acquired  it,  and  for 
that  reason  the  immunity  may  persist  for  months,  and  even  years. 
Grawitz  attempted  to  base  his  views  on  experiments  on  the  acquired 
immunity  against  the  fungus  of  the  lily  of  the  valley,  but  Loeffler2 
soon  demonstrated  that  this  thesis  could  not  be  maintained,  and  that 
the  immunity  assumed  by  Grawitz  did  not,  in  reality,  exist 

It  will  be  seen  that  all  the  theories  summarised  above  are  marked 
by  their  vague  character  and  want  of  precision ;  this  is  not  at  all 
astonishing  when  we  take  into  consideration  the  very  imperfect 
knowledge  of  the  phenomena  of  immunity.  It  is  evident  that  if  we 
wish  to  gain  a  satisfactory  idea  of  the  mechanism  of  the  resistance 
of  the  animal  body  against  pathogenic  micro-organisms,  we  must 
inform  ourselves  as  to  the  modifications  which  take  place  in  the 
organs  and  tissues  at  the  time  of  the  acquisition  of  the  immunity, 
and  also  find  out  what  becomes  of  the  micro-organisms  in  a  refractory 
animal. 

We  have  seen  that  Chauveau  demonstrated  that  anthrax  bacilli 
when  injected  into  the  vessels  of  Algerian  sheep  disappear,  but  he 
was  unable  to  say  anything  as  to  the  way  in  which  this  disappearance 
was  brought  about  in  nature.  Buchner  accepted  the  reinforced 
resistance  of  inflamed  organs  without  being  able  to  describe  the 
phenomena  which  manifest  themselves  during  the  inflammation  of 
tissues  invaded  by  the  pathogenic  micro-organisms. 

Independently  of  these  theoretical  and  rather  speculative  views 
on  immunity,  there  has  been  an  addition  to  our  scientific  assets  of 
fairly  exact  data  on  the  relation  of  certain  pathogenic  organisms  to 
the  organs  and  tissues  of  susceptible  or  refractory  animals.  When,  as 

1  Vir chow's  Archiv,  1881,  Bd.  LXXXIV,  S.  87. 

2  Mitth.  a.  d.  k.  Gsndhtsamte,  Berliu,  1881,  Bd.  I,  S.  134. 

B.  33 


514  Chapter  XVI 

[537]  a  result  of  the  labours  of  Davaine  and  Obermeyer,  the  attention  of 
pathologists,  especially  of  those  working  at  pathological  histology, 
was  drawn  to  the  part  played  by  micro-organisms  in  infective  diseases, 
a  diligent  search  was  instituted  for  these  organisms  in  sections  of 
the  organs  of  persons  who  had  died  from  various  diseases.  Masses 
of  cocci  especially  were  found  in  the  organs  of  individuals  who  had 
died  from  diphtheria,  puerperal  fever,  and  various  forms  of  pyaemia. 
In  the  course  of  these  investigations  attention  was  drawn  fairly  fre- 
quently to  the  presence  of  micro-organisms  inside  the  white  corpuscles 
of  pus  and  of  other  morbid  products.  Amongst  the  first  to  make 
this  observation  I  may  cite  Hayem1  in  France,  and  Birch-Hirschfeld2, 
Klebs,  Rindfleisch,  von  Recklinghausen,  and  Waldeyer  in  Germany. 
Klebs3  speaks  of  the  presence  of  micro-organisms  in  infected  wounds, 
in  the  interior  of  contractile  white  corpuscles,  and  attributes  to 
these  cells  the  principal  r61e  in  the  transport  of  these  parasites  in 
the  lymphatic  tissue.  Waldeyer4  cites  a  case  of  puerperal  fever  in 
which  the  corpuscles  of  the  peritoneal  pus  were  filled  with  bacteria. 
Similar  observations  were  by  no  means  rare ;  and  they  led  to  a 
general  conclusion  that  micro-organisms  meet  with  such  favourable 
conditions  inside  the  leucocytes  that  they  would  contribute  to  their 
dissemination  through  the  body.  This  opinion  had  become  so  general 
that  when  Koch5,  in  frogs  inoculated  with  anthrax  bacilli,  made  the 
discovery  of  round  cells  containing  large  numbers  of  these  micro- 
organisms he  did  not  hesitate  to  conclude  that  the  bacilli  found  a 
favourable  medium  in  the  substance  of  these  elements.  Now  the 
frog,  under  ordinary  conditions,  is  refractory  to  anthrax. 

As  early  as  1874,  however,  Panum6  had  given  expression  to  the 
view,  in  a  vague  fashion  it  is  true,  that  leucocytes  might  assist  in 
the  destruction  of  micro-organisms.  In  his  memoir  on  putrefactive 
poisons  we  find  a  note  wherein  occurs  the  following  reflection : 

[538] "  For  the  solution  of  the  question  as  to  how  and  in  what  situations 
the  ordinary  bacteria  of  putrefaction  disappear,  an  interesting  com- 
munication made  by  Birch-Hirschfeld  seems  to  me  to  furnish  an 

1  Compt.  rend.  Soc.  de  Uol,  Paris,  1870,  p.  115 ;  Gaz.  held,  de  med.,  Paris,  1871, 
p.  291. 

2  Abstract  in  Schmidts  Jahrb.,  Leipzig,  1872,  Bd.  OLX,  S.  97. 
"Beitrage  zur  pathologische  Anatomie  der  Schusswunderi,"  Leipzig,  1872. 

4  Archivf.  Gynaek.,  Berlin,  1872,  Bd.  in,  S.  293. 

6  Cohn's  Beitr.  z.  Biol.  d.  Pflanzen,  Breslau,  1876,  Bd.  n,  S.  300. 

6  Virchow's  Archiv,  1874,  Bd.  LX,  S.  347. 


Historical  sketch  on  Immunity  515 

indication.  According  to  this  observer  the  micrococci,  introduced 
into  the  circulation,  are  deposited  in  the  lymphatic  glands  and  in 
the  spleen,  after  having,  for  the  most  part,  entered  into  the  blood 
corpuscles.  That  the  ordinary  bacilli  of  putrefaction  really  die  in 
the  body  is  proved,  not  only  by  the  circumstance  that  they  remain 
inactive  after  the  acute  paroxysm  of  putrid  intoxication  has  been 
happily  surmounted,  but  also  by  the  important  observations  made  by 
Eberth  on  the  iunocuousness  of  the  inoculation  of  ordinary  bacteria 
into  the  cornea."  These  lines  contain  the  indication  that  the  cor- 
puscles of  the  blood  (in  this  case  undoubtedly  leucocytes)  ingest 
the  bacteria  introduced  in  the  blood  current  and  destroy  them. 

Some  years  later,  in  1877,  Grawitz1,  in  connection  with  his 
researches  on  the  parasite  of  the  lily  of  the  valley,  made  the  remark 
that  the  fungi,  when  introduced  into  the  blood  of  mammals,  are 
seized  by  the  white  corpuscles  and  thus  "  withdrawn  from  contact 
with  the  assimilable  fluid."  Gaule2  who,  as  we  know,  sought  to 
demonstrate  that  the  Drepanidiiim  of  the  frog's  blood  is  nothing 
but  the  fragments  of  cell  nuclei  transformed  into  '  Wurmchen,'  has 
described  the  structure  of  these  organisms  in  the  amoeboid  cells  of 
the  spleen.  "I  happened  on  one  occasion/'  he  writes,  "to  observe 
an  amoebocyte  of  the  spleen  of  the  frog  which  in  a  short  time  in- 
gested three  '  Wiirmchen/  and  then  went  away  briskly  without  leaving 
any  trace  of  where  it  had  been.  Following  its  movements  I  was 
able  at  the  first  to  make  out  within  the  contents  of  the  amoebocyte 
the  refractile  body  of  the  '  Wiirmchen.'  But  this  body  became  paler, 
and  half-an-hour  later  it  had  been  completely  assimilated."  Un- 
doubtedly these  "Wiirmchen"  were  nothing  but  parasites  (Dre- 
panidium),  and  have  no  connection  with  the  cell  nuclei  of  frogs. 
Their  ingestion,  followed  by  destruction,  was,  therefore,  a  defensive 
act  on  the  part  of  the  body  manifested  by  the  amoeboid  cells  of 
the  splenic  pulp. 

In  the  same  year,  1881,  in  which  this  observation  by  Gaule  was 
published,  Roser3,  assistant  in  surgery  at  Marburg,  published  a  small 
pamphlet  on  the  lower  animals.  In  this  pamphlet  the  possibility  of 
growing  certain  unicellular  organisms  in  urine  and  milk  and  the  [539] 
adaptation  of  these  organisms  to  saline  solutions  received  special 
mention.  At  the  end  of  one  of  his  paragraphs  Roser  expresses  his 

1  Virchow's  Archiv,  1877,  Bd.  LXX,  S.  546 ;  1881,  Bd.  LXXXIV,  S.  87. 

2  Archiv  f.  PhysioL,  Leipzig,  1881,  S.  308,  Taf.  V. 

3  "Beitrage  zur  Biologic  niederster  Organisineu,"  Marburg,  1881. 

33—2 


516  Chapter  XVI 

views  on  immunity,  although  this  subject  was  not  discussed  at  all  in 
his  pamphlet.  He  expresses  himself  thus  :  "  The  immunity  of  animals 
and  plants  in  complete  health  depends  in  my  opinion :  (1)  on  the 
relative  quantity  of  salt  contained  in  their  fluids,  and  (2)  on  the 
property  of  their  contractile  cells  of  ingesting  the  enemy  which  enters 
the  animal  body  "  (p.  18).  As  these  statements  have  been  put  forth 
without  receiving  any  further  development,  in  the  midst  of  all  kinds 
of  other  speculations,  it  is  not  astonishing  that  the  words  I  have  just 
quoted,  as  well  as  Roser's  pamphlet  itself,  should  not  have  attracted 
the  attention  of  either  zoologists  or  medical  men.  In  the  reviews  for 
these  two  sciences  (Schmidt's  Jahrbucher  and  the  Zoologischer  Jahres- 
bericht  of  the  Zoological  Station  at  Naples)  it  is  not  even  mentioned. 
It  appears  that  not  only  did  other  biologists  and  medical  men  attach 
no  importance  to  Roser's  speculations,  but  that  the  author  himself 
did  not  claim  any  great  value  for  them.  I  draw  this  conclusion 
from  the  fact  that  five  years  after  his  first  pamphlet  he  published 
a  second  on  inflammation  and  healing1  in  which  he  does  not  apply 
his  theory  of  immunity  to  explain  these  two  phenomena.  This 
new  work  is  of  an  even  more  speculative  character  than  was  the 
first,  and  instead  of  attempting  to  show  any  relation  between  the 
anti-infective  part  played  by  the  leucocytes  and  their  migration 
during  inflammation,  Roser  insists  on  the  fundamental  independence 
of  this  phenomenon  of  healing.  For  him  the  inflammation,  accom- 
panied by  diapedesis,  must  not  be  looked  upon  as  a  healthy  reaction 
of  the  body,  but  as  a  manifestation  of  disease.  The  heat  which  is 
observed  under  these  conditions  must  be  attributed  in  part  at  least 
to  the  production  of  heat  by  infective  micro-organisms.  I  must 
confess  that  Roser's  two  pamphlets  were  unknown  to  me  for  many 
years,  and  it  was  Hueppe  who  drew  my  attention  to  them  by  his 
mention  of  them  in  the  fourth  edition  of  his  work  on  bacterio- 
logical methods2  which  appeared  in  1889.  I  had  then,  independently 
of  the  Marburg  surgeon  and  by  a  totally  different  path,  arrived  at 
my  conclusions  as  to  the  part  played  by  the  amoeboid  cells.  At  the 
commencement  of  my  researches  on  healing  and  immunity  the 
[540]  passages  cited  above  from  the  publications  of  Panum,  Gaule,  and 
Grawitz  were  also  unknown  to  me.  Having  long  studied  the  problem 
of  the  germinal  layers  in  the  animal  series,  I  sought  to  gain  some 
idea  of  their  origin  and  significance.  The  part  played  by  the 

1  Roser,  "  Ueber  Entziindung  und  Heilung,"  Leipzig,  1886. 

2  "Methoden  der  Bacterienforschung,"  4te  Aufl.,  Wiesbaden,  1889,  S.  10. 


Historical  sketch  on  Immunity  517 

ectoderm  and  the  entoderm  appeared  quite  clear,  and  the  former 
might  quite  reasonably  be  regarded  as  the  cutaneous  investment  of 
primitive  multicellular  animals,  whilst  the  latter  might  be  regarded 
as  their  organ  of  digestion.  The  discovery  of  intracellular  digestion 
in  many  of  the  lower  animals  led  me  to  regard  this  phenomenon  as 
characteristic  of  those  ancestral  animals  from  which  might  be  derived 
all  the  known  types  of  the  animal  kingdom  (excepting,  of  course, 
the  Protozoa).  The  origin  and  the  part  played  by  the  mesoderm 
appeared  the  most  obscure.  Thus,  certain  embryologists  supposed 
that  this  layer  corresponded  to  the  reproductive  organs  of  primitive 
animals :  others  regarded  it  as  the  prototype  of  the  organs  of  loco- 
motion. My  embryological  and  physiological  studies  on  sponges  led 
me  to  the  conclusion  that  the  mesoderm  must  function  in  the  hypo- 
thetically  primitive  animals  as  a  mass  of  digestive  cells,  in  all  points 
similar  to  those  of  the  entoderm.  This  hypothesis  necessarily  attracted 
my  attention  to  the  power  of  seizing  foreign  corpuscles  possessed  by 
the  mesodermic  cells.  This  fact  has  long  been  recognised.  It  was 
known  that  the  white  corpuscles  of  the  Yertebrata  often  contained 
various  kinds  of  cells,  especially  red  and  white  blood  corpuscles.  It 
was  known,  also,  that  the  amoeboid  cells  were  capable  of  ingesting 
granules  of  coloured  substances.  When  making  an  injection  of 
indigo  into  the  vessels  of  Thetys,  Haeckel1  in  1858  was  surprised  to 
find  the  blue  granules  inside  the  amoeboid  blood  corpuscles  of  this 
beautiful  gasteropod  mollusk.  This  fact  has  since  been  confirmed  by 
many  observers,  and  the  capacity  of  the  amoeboid  cells  to  take  up 
foreign  bodies  became  recognised  as  a  general  phenomenon.  Never- 
theless this  phenomenon  was  not  regarded  as  being  analogous  to 
digestion.  Thus  Haeckel2  himself,  in  his  researches  on  the  calcareous 
sponges,  advocated  the  view  that  the  foreign  bodies  penetrated 
into  the  interior  of  the  viscous  protoplasm  in  a  purely  passive 
fashion. 

Observations  that  I  made  on  sponges  and  on  certain  pelagic  [511] 
animals,  transparent  and  of  simple  organisation,  convinced  me  that 
the  presence  of  foreign  corpuscles  in  the  amoeboid  cells  of  the  meso- 
derm must  be  attributed  to  an  active  ingestion  by  these  cells  which, 
in  every  respect,  might  be  compared  to  the  phenomena  of  intra- 
cellular digestion  in  the  epithelial  cells  of  the  digestive  canal  of 
many  of  the  lower  animals.  In  order  to  demonstrate  this  fact 

1  "  Die  Radiolarien,"  Berlin,  1862. 

2  "  Die  Kalkschwamme,"  Berlin,  1872. 


518  Chapter  XVI 

clearly  it  was  necessary  to  bring  forward  exact  experimental  proof. 
I  set  myself,  therefore,  during  my  stay  at  Messina  in  1882  and 
1883,  to  study  the  role  of  the  amoeboid  cells  of  the  mesoderm 
from  the  point  of  view  of  intracellular  digestion.  I  found  it  an 
easy  matter  to  demonstrate  that  these  elements  seized  foreign  bodies 
of  very  varied  nature  by  means  of  their  living  processes,  and  that 
certain  of  these  bodies  underwent  a  true  digestion  within  the  amoeboid 
cells.  My  principal  thesis,  that  is  to  say  the  idea  of  the  intimate 
relations  between  the  entoderm  and  the  mesoderm,  was  thus  fully 
confirmed. 

Pondering  over  these  results,  which  were  quite  new  at  the  time, 
the  idea  suggested  itself  to  me  that  the  digestive  function,  so  pro- 
foundly rooted  in  the  mesodermic  elements,  must  play  a  part  in  many 
of  the  vital  phenomena  of  animals.  Starting  from  this  standpoint,  I 
succeeded  in  demonstrating  that,  during  the  very  complicated  meta- 
morphoses of  Echinoderms,  such  as  the  Synaptae,  the  amoeboid  cells 
of  the  mesoderm  fulfil  a  function  in  the  atrophy  of  numerous  larval 
organs.  I  have  never  prosecuted  any  medical  studies;  but  some 
time  before  my  departure  for  Messina  I  listened  to  the  reading  of 
Cohnheim's  treatise  on  General  Pathology,  and  I  was  struck  by  his 
description  of  the  facts  and  of  his  theory  of  inflammation.  The 
former,  especially  his  description  of  the  diapedesis  of  the  white 
corpuscles  through  the  vessel  wall,  seemed  to  be  of  momentous 
interest.  His  theory,  on  the  other  hand,  appeared  to  be  extremely 
vague  and  nebulous.  It  occurred  to  me  that  a  comparative  study  of 
inflammation  in  lower  animals  of  simple  organisation  would  certainly 
throw  light  on  the  very  complex  pathological  phenomena  in  the 
Vertebrata,  even  in  the  frog  which  had  served  as  the  starting-point 
for  Cohnheim's  remarkable  experiments. 

Since,  in  the  atrophy  of  the  larval  organs  of  the  Synaptae,  the 
essential  role  is  accomplished  by  the  amoeboid  cells  of  the  mesoderm 
which  accumulate  and  unite  into  masses,  the  richness  of  inflammatory 
exudations  in  white  corpuscles  may  perhaps  signify  that  these  cor- 
[542]  puscles  have  a  very  important  function  to  fulfil.  This  reflection  led  me 
to  make  the  following  experiment :  to  wound  and  introduce  spines  be- 
neath the  skin  of  very  transparent  marine  animals  ;  if  my  hypothesis 
should  be  well  founded  this  should  bring  about  an  accumulation  of 
amoeboid  cells  at  the  injured  spot.  I  selected  for  this  purpose  the 
large  Bipinnaria  larvae  of  star-fish,  so  abundant  at  Messina,  and 
inserted  prickles  of  the  rose  into  their  bodies.  Very  shortly  these 


Historical  sketch  on  Immunity  519 

prickles  were  found  to  be  surrounded  by  a  mass  of  amoeboid  cells 
such  as  we  see  in  human  exudation  as  the  result  of  the  introduction 
of  a  spine  or  other  foreign  body.  The  whole  process  took  place 
under  my  eyes  in  a  transparent  animal  possessing  neither  blood  nor 
other  vessels,  nor  a  nervous  system.  The  first  point  was  settled. 
The  inflammatory  exudation  must  be  considered  as  a  reaction  against 
all  kinds  of  lesions,  the  exudation  being  a  more  primitive  and  more 
ancient  phenomenon  in  inflammation  than  are  the  functions  of  the 
nervous  system  or  of  the  vessels. 

I  know  quite  well  that,  at  the  period  when  I  made  my  researches 
(1882),  pathologists  regarded  inflammation  as  the  consequence,  if  not 
always,  at  least  in  the  majority  of  cases,  of  the  penetration  of  micro- 
organisms. From  this  followed  the  conclusion  that  the  diapedesis 
and  accumulation  of  white  corpuscles  in  inflammatory  diseases  must 
be  regarded  as  modes  of  defence  of  the  organism  against  micro- 
organisms, the  leucocytes  in  this  struggle  devouring  and  destroying 
the  parasites.  According  to  this  hypothesis  the  significance  of  in- 
flammation at  once  became  simple  and  clear.  With  the  object  of 
verifying  my  hypothesis  I  began  to  make  experiments  on  the  lower 
animals,  so  abundant  in  the  Straits  of  Messina,  and  to  make  myself 
acquainted  with  the  results  that  had  been  obtained  in  general 
pathology  and  in  pathological  histology.  A  perusal  of  Ziegler's 
treatise  on  Pathological  Anatomy  made  it  clear  to  me  that  in  these 
branches  of  medical  science  there  had  long  been  accumulated  a  great 
number  of  observations  fitted  to  facilitate  the  acceptation  of  the  new 
hypothesis  on  inflammation  and  healing.  Numerous  and  well- 
established  facts  on  the  absorption  of  extravasated  blood,  on  the 
fate  of  the  coloured  corpuscles  in  the  body,  on  the  presence  of 
micro-organisms  inside  leucocytes,  etc.,  confirmed  me  in  my  view. 

When  I  had  got  together  certain  information  and  a  number  of 
facts  in  support  of  my  hypothesis  I  communicated  the  results  to  my 
lamented  friend,  Kleinenberg,  at  that  time  Professor  in  the  University 
of  Messina.  Both  medical  man  and  zoologist,  he  was  well  qualified  [543] 
to  offer  a  judgment  upon  the  matter  ;  this  judgment  was  favourable. 
Sometime  later  I  had  the  great  pleasure  of  meeting  the  celebrated 
Professor  Virchow  at  Messina.  I  imparted  to  him  my  ideas  and  he 
was  kind  enough  to  come  with  me  to  examine  my  preparations  of 
Bipinnaria  larvae  and  other  lower  animals  in  which  I  had  set  up  the 
phenomena  of  inflammation  without  the  assistance  of  nervous  or 
vascular  systems.  This  eminent  observer  greatly  encouraged  me  to 


520  Chapter  XVI 

continue  my  investigations.  When  I  explained  to  him  my  view  that 
the  inflammatory  reaction  on  the  part  of  the  amoeboid  cells  could 
only  be  understood  by  accepting  the  hypothesis  that  the  white 
corpuscles  gave  chase  to  the  micro-organisms  and  destroyed  them, 
Virchow  replied  that  in  pathology  just  the  opposite  was  invariably 
taught.  The  general  opinion  was  that  micro-organisms  were  certainly 
found  inside  the  leucocytes  and  that  they  made  use  of  these  cells  as 
a  means  of  transport  and  of  dissemination  through  the  body. 

During  my  stay  at  Messina  my  researches  were  limited  to  the 
lower  animals,  but  later  I  began  to  study  inflammation  and  the 
phenomena  of  infection  in  the  Vertebrata.  It  was  not  until  eight 
months  after  I  had  commenced  my  researches  in  this  direction  that  I 
decided  to  publish  my  results.  I  first  set  them  forth  in  an  address 
given  at  Odessa  before  the  Congress  of  Naturalists  and  Medical  Men 
in  1883.  Later,  they  were  published  in  a  special  article  inserted  in 
Qaus's  Arbeiten  at  Vienna1,  and  in  a  small  work  which  appeared  in 
the  Biologisches  Centralblatt2.  I  sought  especially  to  develop  the 
idea  that  the  intracellular  digestion  of  unicellular  organisms  and  of 
many  Invertebrata  had  been  hereditarily  transmitted  to  the  higher 
animals  and  retained  in  them  by  the  amoeboid  cells  of  mesodermic 
origin.  These  cells,  being  capable  of  ingesting  and  digesting  all 
kinds  of  histological  elements,  may  apply  the  same  power  to  the 
destruction  of  micro-organisms.  In  order  to  support  this  conclusion 
I  introduced  various  kinds  of  bacteria  into  the  bodies  of  some  of  the 
lower  animals  and  I  demonstrated  that  they  were  ingested  and 
destroyed  by  the  amoeboid  cells.  It  was  evident,  however,  that  this 
proof  was  not  sufficient.  I  then  set  myself  to  study  the  diseases  of 
small  Invertebrata  sufficiently  transparent  to  be  observed  directly 
under  the  microscope.  The  Daphniae,  those  small  Crustacea  so 
numerous  and  so  frequent  in  fresh  water,  furnished  me  with  a  favour- 
[544]  able  medium  in  which  to  study  a  real  struggle  which  takes  place 
between  their  leucocytes  and  the  spores  of  a  vegetable  parasite 
belonging  to  the  group  of  the  Blastomycetes.  In  many  cases  the 
amoeboid  cells  guarantee  the  integrity  of  the  animal  by  devouring  a 
large  number  of  these  spores  and  transforming  them  into  an  inert 
detritus.  In  other  cases,  on  the  contrary,  the  fungi  get  the  upper 
hand  in  the  struggle  ;  they  succeed  in  germinating  and  in  overcoming 
the  resistance  of  the  leucocytes  by  reproducing  themselves  rapidly 

1  Arb.  a.  d.  zool.  Inst.  d.  Univ.  Wien,  1883,  Bd.  v,  S.  141. 

2  Biol.  Centralbl.,  Erlangen,  1883,  Bd.  m,  S.  560. 


Historical  sketch  on  Immunity  521 

and  by  killing  these  cells  with  their  poisons.    The  history  of  this 
disease  and  of  this  struggle  was  published  in  Vir chow's  Archiv1. 

Some  time  afterwards  I  published  in  the  same  journal  my  work 
on  the  anthrax  bacillus2,  in  which  I  attempted  to  demonstrate  that 
in  the  Vertebrata  also  the  invasion  of  pathogenic  micro-organisms 
sets  up  a  desperate  struggle  between  them  and  the  amoeboid  cells. 

In  these  four  works  I  made  use  of  the  term  "phagocytes"  to 
designate  the  amoeboid  cells  capable  of  seizing  and  digesting  the 
micro-organisms  and  other  formed  elements.  To  the  theory  based 
on  this  property  of  the  defensive  cells  I  gave  the  name  of  "  theory  of 
phagocytes." 

I  thought,  as  already  mentioned  above,  that  the  observations  on 
absorption  and  leucocytes,  which  had  been  accumulating  for  years  in 
pathological  histology,  had  sufficiently  paved  the  way  for  a  favourable 
reception  to  the  idea  that  the  amoeboid  cells  are  defensive  elements 
of  the  body  capable  of  guaranteeing  to  it  immunity  and  cure.  In 
this  I  was  mistaken.  It  was  precisely  the  specialists  in  this  branch  of 
science  who  from  the  first  manifested  the  most  lively  opposition  to 
this  theory. 

However,  in  the  Presidential  Address  delivered  before  the  66th 
meeting  of  the  British  Association  held  at  Liverpool  in  1896,  Lord 
Lister  said3 :  "  If  ever  there  was  a  romantic  chapter  in  pathology,  it 
has  surely  been  that  of  the  story  of  phagocytosis."  These  words 
encourage  me  to  put  before  the  reader  the  essential  features  of  this 
story. 

My  first  two  memoirs  published  in  1883  did  not  in  any  way 
attract  the  attention  of  the  medical  public.  These  investigations 
had  a  character  that  was  too  zoological  to  be  noticed  by  patholo- 
gists.  But  the  two  following  publications,  in  which  I  treated  of  the  [545] 
Daphnia  disease  and  especially  of  bacterial  anthrax,  immediately 
roused  severe  criticism.  Baumgarten4,  the  well-known  pathologist, 
opened  the  battle  by  the  publication  of  a  review  of  my  researches 
on  phagocytosis.  He  attempted  to  sap  the  basis  of  my  theory,  and 
not  contented  with  d,  priori  arguments,  he  set  his  pupils  to  make 
a  series  of  researches  on  the  fate  of  micro-organisms  in  the  refractory 

1  Virchow's  Archiv,  1884,  Bd.  xcvi,  S.  177. 

2  Virchovfs  Archiv,  1884,  Bd.  xcvii,  S.  502. 

3  Rep.  Brit.  Ass.  Adv.  Sci.,  London,  1896,  p.  26;  Rev.  Scient.,  Paris,  17  Octobre, 
1896,  p.  493. 

4  Berl.  klin.  Wchnschr.,  1884. 


522  Chapter  XVI 

animal.  These  researches  resulted  in  several  theses  for  the  doctor's 
degree  which  sought  to  demolish  every  point  of  the  theory  of 
phagocytosis. 

Later,  Baumgarten1  published  a  long  and  above  all  admirably 
written  analytical  article  entitled:  "Zur  Kritik  der  MetschnikofF- 
schen  Phagocytentheorie,"  in  which,  with  much  talent  and  wit,  he 
attempted  to  demolish  the  bases  and  conclusions  of  the  phagocytic 
theory. 

Baumgarten  regards  the  precise  observations  which  I  had  been 
accumulating  for  some  years  as  incorrect  and  refuted  by  the  observa- 
tions and  experiments  of  his  pupils.  The  arguments  that  I  give  to 
justify  my  theory  are,  according  to  the  same  critic,  contrary  to  logic 
and  to  truth.  If  the  phagocytes  are  really  elements  destined  to 
guarantee  the  integrity  of  the  animal  organism  how  is  it,  asks 
Baumgarten,  that  just  at  the  moment  of  greatest  danger,  when  the 
blood  and  the  tissues  are  invaded  by  the  micro-organisms,  the 
leucocytes  are  conspicuous  by  their  absence  ?  The  answer  that  there 
is  no  predestination  in  the  phagocytosis,  and  that  the  danger  is  the 
greater  the  more  feeble  the  phagocytic  reaction — a  fact  which  is  in 
perfect  harmony  with  the  law  of  causes  and  with  the  principles 
of  the  evolution  of  species  according  to  Darwin's  theory — did  not 
satisfy  my  critic.  He  says :  "  If  the  interpretation  which  Metschnikoff 
gives  of  the  activity  of  the  leucocytes  appears  to  be  rather  the 
product  of  a  rich  imagination  than  the  result  of  the  objective  obser- 
vation of  the  seeker,  it  matters  little  that  his  account  of  the  develop- 
ment of  the  leucocyte  in  what  he  wishes  to  see  in  it  should  be  in 
conformity  with  the  principles  of  the  theory  of  evolution"  (p.  4). 

I  was  able  by  numerous  researches2  to  refute  point  by  point  the 
[546]  objections  based  on  the  work  of  Baumgarten's  pupils,  but  that  did 
not  prevent  him  from  persisting  in  his  negation.  Only,  commencing 
by  writing  long  articles,  he  contented  himself,  later,  with  denying  the 
theory  of  phagocytosis  in  small  annual  notes,  appearing  in  his  reviews 
of  works  on  bacteriology,  which  were  unsupported  either  by  argument 
or  by  any  facts  mentioned  in  his  abstracts. 

Baumgarten's  example  was  followed  by  many  other  pathologists. 
Ziegler,  the  well-known  author  of  a  text-book  on  pathological 
anatomy  that  has  certainly  had  a  wider  circulation  than  any  other 

1  Ztschr.f.  Jdin.  Med.,  Berlin,  1888,  Bd.  xv,  S.  1. 

2  Firchow'8  Archiv,  1888,  Bd.  oxiv,  S.  465 ;   Ann.  de  I'Inst.  Pasteur,  Paris, 
1890,  t  IV,  p.  35. 


Historical  sketch  on  Immunity  523 

work,  vigorously  attacked  the  theory  of  phagocytosis.  As  it  was 
precisely  from  this  treatise  that  I  had  acquired  my  knowledge  of  the 
large  number  of  facts  that  had  accumulated  in  pathological  litera- 
ture on  the  part  played  by  leucocytes  in  resorption,  I  was  persuaded 
that  Ziegler,  who  had  collected  these  statements,  would  be  one  of  the 
first  to  recognise  the  importance  of  phagocytosis  in  inflammation, 
healing,  and  immunity.  But  this  distinguished  pathologist,  in 
several  of  his  publications1,  expressed  himself  very  vigorously 
against  the  phagocytic  theory.  The  intervention  of  these  cells, 
according  to  him,  must  be  purely  accidental  and  their  r61e  in  the 
defence  of  the  body  against  the  micro-organisms  very  insignificant. 
The  better  to  demonstrate  this  thesis  he  caused  his  pupils  to  under- 
take investigations  on  several  infective  diseases,  and  these  young 
observers  all  arrived  at  the  same  result,  that  phagocytosis  has 
nothing  to  do  with  the  struggle  of  the  animal  against  the  anthrax 
bacillus  or  against  the  bacillus  of  symptomatic  anthrax.  It  is  the 
less  necessary  to  enter  into  these  details  now  because  I  have,  in  the 
preceding  chapters,  given  sufficient  proofs  of  the  incorrectness  of  the 
objections  advanced  by  Ziegler's  school.  It  has  been  demonstrated 
most  conclusively  (by  Lubarsch's  researches,  as  well  as  by  many 
other  works)  that  in  anthrax  in  man  phagocytosis,  denied  by  one 
of  Ziegler's  pupils,  is  most  marked.  It  is  likewise  well  known  from 
the  researches  of  RufFer,  Leclainche  and  Valle*e,  as  well  as  from  my 
own  observations,  that  in  symptomatic  anthrax,  in  which  the  phago- 
cytic reaction  is  denied  by  another  of  Ziegler's  pupils,  it  is  a  very 
important  and  highly  developed  feature. 

The  opposition  emanating  from  another  eminent  pathologist, 
Weigert2,  particularly  impressed  me,  because  this  investigator  is 
known  not  only  to  be  an  observer  of  great  accuracy  but  to  possess 
a  mind  of  great  imagination  and  generalising  power.  In  several  t54?] 
papers  he  put  forward  his  utmost  ingenuity  to  demolish  the  phago- 
cytic theory  root  and  branch.  He  would  recognise  neither  the 
importance  of  phagocytosis  in  healing  and  immunity,  nor  the  defen- 
sive function  of  the  giant  cells.  Weigert,  however,  contented  himself 
with  formulating  theoretical  objections,  and  no  works  directed  specially 
against  the  doctrine  of  phagocytosis  have  issued  from  his  laboratory. 
It  must  be  stated,  however,  that  although  there  has  been  such  oppo- 

1  "Lehrb.  d.  pathol.  Anat.,"  Jena,  3te  Aufl.;  Beitr.  z.  path.  Anat.,  Jena,  1889, 
Bd.  v,  S.  419. 

2  Fortschr.  d.  Med.,  Berlin,  1887,  Bd.  v,  S.  732;  Ibid.,  1888,  Bd.  vi,  SS.  83,  809. 


524  Chapter  XVI 

sition  on  the  part  of  certain  of  our  most  eminent  pathologists,  others 
amongst  them  have,  from  the  beginning,  expressed  themselves  in  more 
favourable  terms.  Thus,  Virchow1,  in  an  introductory  article  in  the 
101st  volume  of  his  Archiv,  continued  his  friendly  attitude  with 
regard  to  the  works  on  phagocytic  defence  and  spoke  of  them  as 
opening  up  a  new  field  of  research.  Ribbert2,  in  a  series  of  publi- 
cations, maintained  the  importance  of  the  phagocytes  in  the  resist- 
ance offered  by  the  animal  to  the  aggression  of  micro-organisms, 
and  pointed  out,  especially  in  connection  with  the  diseases  set  up 
by  the  staphylococci,  the  frequency  of  the  ingestion  of  these  parasites 
by  the  leucocytes.  He  insists  specially  on  a  modification  of  the 
phagocytic  reaction,  which  consists  in  the  accumulation  of  white 
corpuscles  around  the  centre  of  microbial  infection.  In  these  cases, 
without  the  occurrence  of  any  real  ingestion  of  the  micro-organisms 
into  the  substance  of  the  phagocytes,  these  organisms  may  have  their 
morbific  manifestation  hindered  by  the  assemblage  of  the  white 
corpuscles.  It  is  needless  to  insist  that  this  act,  which  I  referred 
to  in  my  first  work  in  1883,  constitutes  the  prelude  to  a  true 
phagocytosis  and  is  closely  bound  up  with  this  defensive  phenomenon. 
Another  pathologist,  Hess3,  supports  the  theory  of  phagocytosis  by 
confirmatory  researches  of  great  value. 

The  pathologists  who  were  adversaries  of  the  phagocytic  theory 
combined  their  efforts  to  demolish  it,  without  troubling  themselves 
to  replace  it  by  any  other  theory  of  defence  on  the  part  of  the  body 
which  might  more  easily  be  made  to  accord  with  their  principles 
and  their  statements.  Baumgarten  certainly  tried  to  prove  that 
micro-organisms  perish  in  cases  where  immunity  is  produced  or 
healing  occurs,  not  as  the  result  of  the  phagocytic  reaction  or  of  any 
other  manifestation  on  the  part  of  the  menaced  animal,  but  simply 
"of  themselves"  (von  selbst),  that  is  to  say,  they  have  simply 
accomplished  the  normal  cycle  of  their  existence  and  die  a  natural 
[548]  death,  this  bringing  about  healing  and  immunity.  As  may  be  readily 
understood  he  was  unable  to  bring  forward  the  slightest  evidence 
of  the  correctness  of  this  hypothesis,  which,  I  believe,  has  never  been 
accepted  by  anyone,  nor  even  been  defended  by  its  author.  In  this 
respect  the  attacks  directed  against  the  theory  of  phagocytosis  by 
bacteriologists  have  been  of  a  very  different  character.  Not  content 

1   Virchoufs  Archiv,  1885,  Bd.  ci,  S.  12. 

*  Deutsche  med.  Wchnschr.,  Leipzig,  1890,  S.  690. 

3   Virchow' s  Archiv,  1887,  Bd.  cix,  S.  365. 


Historical  sketch  on  Immunity  525 

with  overturning  this  hypothesis,  these  observers  have  sought  to  build 
upon  its  ruins  new  theories  capable  of  offering  a  better  explanation  of 
the  phenomena  of  immunity.  I  must  here  confess  at  the  outset  that 
these  attacks  have  been  much  more  important  than  those  coming 
from  the  pathologists  and  pathological  anatomists,  and  have  led  to 
discoveries  of  the  greatest  value. 

One  of  Fodor's  experiments1,  one  not  altogether  new,  served  as  the 
point  of  departure  for  much  work  and  for  a  large  series  of  objections 
directed  against  the  phagocytic  theory.  The  Hungarian  investigator 
found  that  the  defibriuated  blood  of  the  rabbit  was  capable  of 
destroying  in  vitro  a  great  number  of  anthrax  bacilli.  From  this 
it  was  concluded  that  the  fluids  of  the  living  body  possessed  a 
bactericidal  power  sufficient  to  explain  the  immunity  against  infective 
micro-organisms.  The  destruction  of  the  anthrax  bacillus  by  defibri- 
nated  blood  was  confirmed  by  a  young  American  investigator  of  great 
talent,  Xuttall2,  who  carried  out  an  important  work  on  this  subject 
in  the  laboratory  and  under  the  direction  of  Fliigge  at  Breslau.  He 
was  able  to  follow  step  by  step,  by  the  observation  of  anthrax  bacilli 
on  the  warm  stage,  their  degeneration  under  the  action  of  the 
defibrinated  blood.  This  destruction  of  the  bacilli  took  place  outside 
the  phagocytes.  The  same  phenomenon  could  be  shown  by  the 
method  of  gelatine  plate  cultures.  The  bacilli,  subjected  to  the 
influence  of  the  defibrinated  blood  of  rabbits  and  other  vertebrates, 
usually  died  or  were  markedly  injured.  The  blood  when  heated 
to  55°  C.  completely  lost  its  bactericidal  power. 

These  observations,  perfectly  exact  in  every  detail,  gave  Fliigge3 
and  his  assistant  Bitter4  the  opportunity  to  criticise  vigorously  the 
theory  of  phagocytosis.  The  cells  were  said  to  be  incapable  of  [549] 
ingesting  living  micro-organisms ;  these  latter  must  be  previously 
destroyed  by  the  bactericidal  action  of  the  body  fluids,  and  it  was  only 
their  dead  bodies  which  were  devoured  by  the  phagocytes. 

Fliigge  based  his  criticism  upon  considerations  of  a  general 
character  and  upon  observations  made  mainly  by  Nuttall.  "  There  is 
no  necessary  point  of  analogy,"  says  the  learned  Breslau  hygienist, 
"between  the  ingestion  of  food  and  the  struggle  against  infective 

1  Deutsche  med.  Wchnschr.,  Leipzig,  1886,  S.  617;  Arch.  f.  Hyg.,  Munchen 
u.  Leipzig,  1886,  Bd.  rv,  S.  129. 

-  Ztschr.f.  Hyg.,  Leipzig,  1888,  Bd.  iv,  S.  353. 
3  Ztschr.f.  Hyg,,  Leipzig,  1888,  Bd.  TV,  S.  223. 

*  Ztschr.f.  Hyg.,  Leipzig,  1888,  Bd.  iv,  S.  318. 


526  Chapter  XVI 

micro-organisms,  nor  between  nutritive  substances  and  living  micro- 
organisms" (p.  225).  "From  NuttalPs  results  it  must  evidently  be 
accepted  as  possible  that  the  phagocytes  can  ingest  dead  bacteria 
only  and  that  they  have  not  the  power  of  ridding  the  body  of  the 
living  infective  agents  "  (p.  226).  The  following  passage  is  especially 
significant.  "When  we  examine,  with  an  open  mind,  a  series  of 
preparations  which  show  the  relations  between  the  phagocytes  and 
the  bacteria  in  various  infective  diseases,  the  phagocytes  sometimes 
present  themselves  as  the  victims  of  the  bacteria,  which  continue 
their  triumphal  march ;  sometimes  they  produce  the  impression  of 
tombstones  lying  in  large  numbers  behind  the  line  of  battle  and  after 
the  end  of  the  struggle.  On  the  other  hand,  they  in  no  way  force 
themselves  upon  our  notice  as  instruments  of  slaughter  which  the 
attacked  organism  makes  use  of  to  defend  itself7'  (p.  227). 

These  arguments  have  been  regarded  by  many  investigators  in  all 
countries  as  perfectly  sufficient  to  overthrow  the  phagocytic  theory. 
The  bactericidal  power  of  the  body  fluids  became  the  rallying  cry 
of  a  great  number  of  works  always  directed  to  the  same  object : 
to  replace  the  role  of  phagocytosis  by  that  of  a  bactericidal  power 
of  the  body  fluids.  It  is  quite  unnecessary  to  weary  the  reader  with 
a  list  of  the  very  numerous  publications  that  have  appeared  on  this 
subject  in  every  European  language.  But  it  is  not  possible  to  pass 
over  in  silence  the  work  of  some  of  the  principal  partisans  of  the 
humoral  theory  of  immunity. 

The  first  place  amongst  these  works  certainly  belongs  to  von 
Behring's  memoir1  on  the  natural  immunity  of  white  rats  against 
anthrax.  As  already  stated  in  Chapter  vi  of  this  work,  von  Behring 
discovered  the  very  remarkable  power  possessed  by  the  rat's  blood 
[550]  of  destroying  anthrax  bacilli  with  very  great  rapidity.  This  inves- 
tigator did  not  hesitate  to  conclude  therefrom  that  this  bactericidal 
property  of  the  blood  must,  in  the  rat,  bring  about  a  great  resistance 
against  anthrax.  We  should  have  in  this  case,  then,  an  example  in 
which  the  immunity  did  not  depend  in  any  way  upon  phagocytosis, 
but  would  be  bound  up  entirely  in  a  purely  humoral  property. 

With  the  object  of  deciding  whether  the  bactericidal  property  of 
the  blood  is  really  the  general  and  essential  cause  of  natural  or 
acquired  immunity,  von  Behring,  in  collaboration  with  Nissen2,  carried 
out  a  long  series  of  experiments,  the  results  of  which,  however,  did 

1  CentralU.f.  klin.  Med.,  Bonn,  1888,  No.  38. 

2  Ztschr.f.  Hyg.,  Leipzig,  1890,  Bd.  vin,  S.  412. 


Historical  sketch  on  Immunity  527 

not  confirm  their  expectations.  They  found  that  in  animals  well 
vaccinated  against  certain  bacteria  (notably  Gamaleia's  vibrio  or 
V.  metschnil'ovi),  the  blood  plasma  undoubtedly  acquires  a  high 
specific  bactericidal  power,  but  at  the  same  time  they  satisfied 
themselves  that  the  blood,  even  of  well  immunised  animals,  was 
generally  incapable  of  killing  the  micro-organisms.  The  bactericidal 
property,  then,  according  to  their  researches,  presented  itself  not  as 
a  general  character  but  as  one  of  limited  importance.  These  facts 
even  led  von  Behring  to  abandon  the  theory  of  the  bactericidal 
power  of  the  body  fluids  as  an  explanation  of  immunity. 

This  theory  found  many  warm  partisans,  especially  at  Munich. 
Emmerich  had  already  announced  at  the  International  Congress  of 
Hygiene,  held  at  Vienna  in  1887,  that  in  the  blood  of  rabbits 
vaccinated  against  the  bacillus  of  swine  erysipelas  an  antiseptic 
substance  of  remarkable  activity  is  produced.  To  this,  exclusively, 
in  this  instance,  and  not  to  the  phagocytes,  he  attributed  the  acquired 
immunity.  Later,  Emmerich1  in  an  investigation  carried  out  in 
collaboration  with  di  Mattel  developed  this  view.  We  may  refrain 
from  giving  any  account  of  the  contents  of  their  memoir  as  well  as 
from  criticising  their  conclusions,  as  this  has  already  been  done  in 
Chapter  ix.  Let  us  content  ourselves  with  stating  that  our  own  ex- 
periments, as  well  as  those  made  later  by  Mesnil,  have  demonstrated 
the  inaccuracy  of  Emmerich's  statements. 

Another  Munich  bacteriologist,  H.  Buchner,  at  first  expressed 
himself2  very  favourably  on  the  theory  of  phagocytosis.  He 
regarded  it  as  more  capable  of  explaining  most  of  the  phenomena 
of  immunity  than  was  his  own  older  local  theory.  But  little  by 
little  he  declared  himself  in  formal  opposition  to  the  cellular  theory 
of  immunity  and  went  over  to  the  camp  of  his  sometime  adver-[55i] 
saries.  He  adopted3  the  humoral  theory  of  the  bactericidal  action 
of  the  body  fluids,  upon  which  subject  he  carried  out  several 
important  investigations.  He  was  able  without  difficulty  to  confirm 
Xuttall's  discovery  of  the  disappearance  of  the  microbicidal  power 
when  the  defibrinated  blood  was  heated  to  55°  C.,  and  he  added  to 
this  fundamental  fact  many  others  of  great  value.  He  demonstrated 
the  part  played  by  the  salts  in  the  exercise  of  this  bactericidal  power, 
and  laid  great  stress  on  the  fact  that  this  power  depends  on  the 

1  Fortschr.  d.  Mecl.,  Berlin,  1887,  Bd  v,  S.  653. 

2  Miinchen.  med.  Wchnschr.,  1887. 

8  Centralblf.  Bakteriol.  u.  Parasitenk.,  Jena,  1891,  Bd.  x,  S.  727. 


528  Chapter  XVI 

presence  of  a  special  substance  of  albuminoid  nature,  to  which  he  gave 
the  name  of  alexin.  Buchner1  combatted  with  success  the  idea  that 
I  had  expressed,  according  to  which  the  bactericidal  power  of  the 
body  fluids  is  reduced  in  great  part  to  a  plasmolytic  action  of  the 
blood  serum  upon  certain  micro-organisms.  It  cannot  be  denied  that 
my  hypothesis  is  only  very  partially  applicable,  and  that  the  larger 
share  in  the  bactericidal  action  of  the  body  fluids  belongs  to  the 
alexins.  Buchner  also  made  the  study  of  this  action  more  easy  by 
the  demonstration  that  the  red  blood  corpuscles  of  a  foreign  species 
undergo,  under  the  action  of  the  blood  and  of  the  serums,  a  globu- 
licidal  action  comparable  to  that  which  occurs  in  the  case  of  micro- 
organisms. 

Whilst  Fliigge,  von  Behring  and  many  others  of  the  old  partisans 
of  the  bactericidal  theory  of  the  body  fluids  abandoned  it  more  or 
less  completely  as  an  explanation  of  immunity,  Buchner  remained 
faithful  to  it  and  tried,  aided  by  the  collaboration  of  his  pupils,  as  far 
as  possible  to  defend  it. 

In  France  this  humoral  theory  was  adopted  chiefly  by  Bouchard2 
and  his  pupils,  amongst  whom  I  must  cite  more  particularly  Charrin 
and  Roger.  They  sought  to  confirm  it  by  personal  researches,  the 
greater  part  of  which  were  carried  out  upon  the  bacillus  of  blue 
pus.  These  investigators  studied  it  especially  in  relation  to  acquired 
immunity.  A  comparison  of  the  mode  of  development  of  the  pyo- 
cyanic  bacillus  in  the  serum  of  susceptible  animals  and  of  vaccinated 
animals  of  the  same  species,  convinced  them  of  the  great  importance 
of  the  action  of  the  body  fluids.  In  cases  where  these  fluids  were 
found  to  be  incapable  of  killing  the  micro-organisms  they  exerted 
over  them  an  injurious  influence,  either  by  attenuating  their  virulence, 
[552]  or  by  producing  more  or  less  important  modifications  in  their  forms 
and  functions.  The  essential  cause  of  natural  or  acquired  immunity 
was  always  attributed  by  Bouchard's  school  to  the  property  of  the 
body  fluids.  The  phagocytes  were  said  to  intervene  only  secondarily, 
either  to  carry  off  the  dead  bodies  of  the  micro-organisms,  or  to 
ingest  the  bacteria,  rendered  inoffensive  by  the  humoral  action. 

The  humoral  theory  of  immunity,  with  some  slight  modifications, 
spread  very  generally  into  every  country,  and  many  investigators 
accepted  it  without  reserve.  But  certain  observers  ventured  to  run 
counter  to  the  general  current  and  raised  objections  of  principle 

1  Centralblf.  Bakteriol.  u.  Pamsitenk.,  Jena,  1890,  Bd.  vin,  S.  65. 

2  "Les  microbes  pathogenes,"  Paris,  1892. 


Historical  sketch  on  Immunity  529 

against  the  theory  of  the  bactericidal  power  of  the  fluids  of  the 
body.  After  the  principal  facts  established  by  the  partisans  of  this 
theory  had  been  confirmed,  it  was  asked  whether  the  phenomena 
of  the  destruction  of  micro-organisms  observed  in  vitro  are  really 
equivalent  to  those  produced  in  the  refractory  animal.  A  glance 
at  the  data  brought  together  with  so  much  zeal  was  sufficient  to 
demonstrate  that  this  parallelism  does  not  exist  The  blood  of 
animals  susceptible  to  certain  micro-organisms  was  found  to  be 
bactericidal  for  these  organisms,  whilst  that  of  refractory  animals  was 
incapable  of  destroying  them.  It  is  useless  to  cite  examples,  so 
numerous  are  they.  On  the  other  hand,  the  bactericidal  power  of 
the  body  fluids,  so  marked  for  certain  pathogenic  organisms  such  as 
the  anthrax  bacillus  and  especially  the  cholera  vibrio  and  the  typhoid 
coccobacillus,  is  insignificant  or  nil  as  regards  many  bacteria  against 
which  refractory  animals  are  not  wanting. 

All  these  facts  throw  doubt  on  the  predominating  part  played  in 
immunity  by  the  bactericidal  power  of  the  body  fluids.  Lubarsch1 
attacked  the  humoral  theory,  showing  by  a  great  number  of  experi- 
ments that  animals  whose  fluids  are  very  bactericidal  in  vitro  are 
very  susceptible  to  a  much  smaller  quantity  of  bacteria  of  the  same 
species  introduced  into  the  body.  Thus,  the  defibrinated  blood  and 
the  blood  serum  of  rabbits  destroy  a  large  number  of  bacteria  in 
a  very  short  time,  whilst  the  rabbits  themselves  contract  fatal 
anthrax  after  the  introduction  of  a  small  number  of  these  micro- 
organisms into  the  blood  vessels.  This  contradiction  cannot  be\[553] 
explained  except  by  the  profound  changes  which  the  blood  must] 
undergo  outside  the  body.  Facts  of  the  same  nature  have  been| 
shown  for  the  anthrax  of  rats  by  Hankin,  Roux,  and  ourselves,  as 
described  in  Chapter  VL 

The  International  Congress  of  Medicine,  assembled  at  Berlin  in 
1890,  was  the  first  occasion  on  which  I  spoke  publicly  of  the 
new  theories  of  immunity.  In  the  addresses  given  at  the  general 
meetings,  leaders  of  medical  science  in  several  countries  summed  up 
their  opinion  on  this  question.  Koch2,  in  his  memorable  report, 
declared  that  the  new  acquisitions  had  destroyed  the  basis  of  the 
theory  of  phagocytes,  and  that  consequently  it  must  give  place 
to  the  humoral  theory  of  immunity.  Bouchard  took  up  a  more 
conciliatory  position,  but,  according  to  him,  the  bactericidal  power  of 

1  Centralblf.  Bakteriol.  u.  Parasitenk.,  Jena,  1889,  Bd.  vi,  SS.  481,  529. 

2  "  Ueber  bacteriologische  Forschung,"  Berlin,  1890. 

B.  34 


530  Chapter  XVI 

the  fluids  of  the  body  was  the  primary  and  essential  cause  of  immunity. 
The  phagocytes  only  intervened  later,  in  order  to  finish  the  work 
begun  without  their  assistance.  Lord  Lister  expressed  himself1,  on 
the  other  hand,  much  more  favourably  on  the  subject  of  the  theory 
of  phagocytosis.  This  observer,  who  is  not  only  a  great  surgeon, 
but  is  perhaps  even  more  remarkable  for  his  great  powers  of 
generalisation,  has  paid  special  attention  to  the  problem  of  immunity. 
With  the  object  of  clearing  up  this  very  complicated  and  at  the  same 
time  important  question,  Lord  Lister  seized  the  occasion  of  the 
meeting  of  the  International  Congress  of  Hygiene  in  London  in 
1891,  to  bring  about  an  exchange  of  views  between  the  partisans 
of  the  various  theories  of  immunity.  Under  his  presidency  he 
devoted  an  entire  sitting  of  the  Section  of  Bacteriology  to  the 
discussion  of  this  question.  Buchner  presented  a  report2  drawn  up 
exclusively  from  the  point  of  view  of  the  humoral  theory  and  devoted 
to  the  demonstration  of  the  slight  importance  of  phagocytosis,  and 
also  to  the  preponderant  part  played  by  the  alexins  dissolved  in  the 
body  fluids  and  circulating  in  the  plasma  of  the  blood.  He  attempted 
to  harmonise  the  facts  on  the  bactericidal  power  of  serums  observed 
in  vitro  with  the  special  conditions  to  be  met  with  in  the  animal 
body.  He  specially  insisted  on  the  point  that,  in  the  blood  and  the 
organs,  the  alexins  cannot  act  with  the  same  rapidity  that  they  can 
[554]  in  test-tubes  containing  serum.  In  this  way  he  recognised  that 
between  the  bactericidal  action  in  vitro  and  that  in  the  body  of  the 
animal,  there  exists  a  marked  difference,  but  he  would  not  consent 
to  attribute  it  in  the  latter  case  to  the  intervention  of  the  phagocytes. 
Roux3  also  made  a  report  on  immunity  at  the  same  sederunt, 
speaking  very  distinctly  in  favour  of  the  cellular  theory.  A  chemist 
by  inclination,  he  was  sympathetic  at  first  to  the  humoral  theories  of 
immunity.  Working  with  Pasteur,  and  side  by  side  with  him,  Roux, 
from  the  beginning  of  the  new  era  of  medical  science,  had  made 
numerous  experiments  on  the  part  played  by  the  body  fluids  in 
immunity.  But  as  the  results  were  not  sufficiently  precise  and 
demonstrative  they  were  soon  abandoned.  The  attachment  of  Roux, 
however,  to  the  humoral  theories  was  manifested  in  his  work,  carried 
out  in  part  with  Chamberland4,  on  the  subject  of  vaccination  by 

"  The  present  position  of  antiseptic  surgery,"  Berlin-,  1890. 

2  Munchen.  med.  Wchnschr.,  1891,  SS.  551,  574. 

3  Ann.  de  VInst.  Pasteur,  Paris,  1891,  t.  V,  p.  517. 

4  Ann.  de  VInst.  Pasteur,  Paris,  1887, 1. 1,  p.  561. 


Historical  sketch  on  Immunity  531 

means  of  microbial  products.  Later,  having  obtained  a  deeper  know- 
ledge of  various  facts  concerning  natural  and  acquired  immunity, 
he  rallied  to  the  cellular  conception  and  developed  it  in  his  report 
presented  to  the  above  Congress  in  London.  Several  microbiologists 
took  part  in  the  discussion,  and  I  myself1  was  able  to  communicate 
certain  facts  concerning  the  immunity  of  guinea-pigs,  acquired  as  the 
result  of  vaccination  against  Gamaleia's  vibrio.  I  chose  this  example 
because  it  presented,  according  to  von  Behring  and  Nisseu,  the 
clearest  case  of  a  bactericidal  property  developed  during  the  course 
of  immunisation.  I  was  able  to  furnish  the  proof  that,  in  the 
vaccinated  animal,  the  micro-organism  in  question,  in  spite  of  the 
great  bactericidal  power  of  the  blood  serum  in  vitro,  remains  alive 
in  the  animal  body  for  a  long  time,  and  that  its  destruction  is  effected 
by  the  phagocytes,  which  ingested  it  alive.  In  this  example  I  showed 
that  the  leucocytes  of  the  exudation,  that  have  ingested  vibrios,  may 
still  furnish  cultures  of  this  organism  if  they  are  taken  from  the  body 
and  transferred  in  hanging  drop  to  the  incubator. 

The  fact  that,  even  in  the  case  which  appeared  most  to  favour 
the  humoral  conception  of  acquired  immunity,  phagocytes  play  the 
principal  part,  must  to  many  members  of  the  Congress  have  appeared 
sufficiently  significant.  Indeed,  several  observers  who  were  present 
at  the  debates,  received  the  impression  that  the  phagocytic  theory 
had  not  been  overturned  by  its  adversaries.  At  this  period  the  [555] 
question  of  the  importance  of  antitoxins  from  the  point  of  view 
of  immunity  had  scarcely  been  raised.  The  great  discovery  made  by 
von  Behring  and  Kitasato  was  already  accepted  by  everyone;  but 
there  was  no  ground  for  attributing  to  it  any  general  importance. 
In  fact,  though  proved  for  tetanus  and  diphtheria,  and  extended  by 
Ehrlich's  beautiful  experiments  to  the  vegetable  toxins  (ricin,  abrin, 
and  robin),  the  antitoxic  property  of  the  fluids  of  the  body  presented 
itself  rather  as  a  special  than  as  a  general  phenomenon.  It  is  in  this 
sense  that  Roux  had  assigned  to  it  its  place  in  the  chapter  of 
immunity.  The  two  diseases,  against  which  antitoxic  serums  had  been 
discovered,  are  certainly  distinguished  from  the  great  majority  of 
infections  by  the  localisation  of  the  micro-organisms  and  the  abundant 
secretion  of  their  toxins. 

It  was  only  after  the  London  Congress  that  this  question  came 
prominently  forward.  Yon  Behring  thought  that  the  antitoxic  power 
of  the  body  fluids  is  generally  distributed  in  all  cases  of  acquired 

1  Ann.  de  VInst.  Pasteur,  Paris,  1891,  t.  v,  pp.  465,  534. 

34—2 


532  Chapter  XVI 

immunity,  and  that  micro-organisms,  introduced  into  the  animal 
possessing  this  power,  become  incapable  of  any  pathogenic  manifesta- 
tion. Certain  facts,  brought  together  in  Bouchard's  laboratory,  tell 
against  the  hypothesis  I  have  just  mentioned.  With  the  object  of 
throwing  light  on  this  question  I  began,  immediately  after  the  close 
of  the  Congress,  to  study  the  acquired  immunity  of  rabbits  against 
the  micro-organism  of  the  pneumo-enteritis  of  pigs.  I  was  able 
to  demonstrate1  that  in  this  case  the  resistance  of  the  animal  against 
j  the  micro-organisms  does  not  depend  on  the  acquisition  of  any  anti- 
! toxic  property  by  the  body  fluids;  such  a  property  is  completely 
absent.  At  the  same  time  I  showed  that  the  serum  of  vaccinated 
rabbits  possesses  a  very  marked  protective  power  against  infection  by 
the  coccobacillus  of  pneumo-enteritis.  It  was  for  the  first  time  proved 
that  independently  of  the  antitoxic  and  bactericidal  properties  of 
serums,  there  exists  another  special  property,  the  anti-infective 
property.  This  I  conceived  to  be  of  the  nature  of  a  stimulant  action 
on  the  part  of  the  phagocytes. 

It  has  already  been  stated  in  an  earlier  chapter  that  before  the 
discovery  of  antitoxins  Richet  and  Hericourt2  had  observed  an 
immunising  action  of  the  serum  of  animals  refractory  to  staphylococci. 
These  observers  were  content  with  this  demonstration  and  did  not 
seek  to  penetrate  more  deeply  into  the  mechanism  of  the  action 
of  their  serum.  For  this  reason  when  von  Behring  and  Kitasato 
[556]  announced  their  discovery  of  antitoxic  serums  it  was  generally 
thought  that  the  antistaphylococci  serums  were  also  antitoxic  serums. 
The  immunity  against  the  micro-organism  of  the  pneumo-enteritis  of 
pigs  taught  us  that  here  we  might  have  to  deal  with  quite  a  different 
matter.  It  was  soon  demonstrated  that  the  serum  from  the  immunised 
animal  might  in  fact,  without  being  antitoxic,  present  the  same  anti- 
infective  property  as  in  the  case  of  pneumo-enteritis.  That  was  first 
proved  in  the  case  of  the  experimental  disease  set  up  by  Koch's 
cholera  vibrio. 

The  reappearance  of  cholera  in  Europe  in  1892  drew  the  attention 
of  bacteriologists  to  this  disease,  and  was  the  occasion  of  many  new 
researches  on  immunity  against  the  cholera  vibrio.  Several  important 
works  on  this  question  were  published  by  Pfeiffer3,  at  this  period 
director  of  the  scientific  staff  of  the  Koch  Institute  at  Berlin.  He 

1  Ann.  de  FInst.  Pasteur,  Paris,  1892,  t.  vi,  p.  289. 

1  Cvm.pt.  rend.  Acad.  d.  sc.,  Paris,  1888,  t.  cvn,  pp.  690,  748. 

3  Ztschr.f.  Hyg.,  Leipzig,  1894,  Bd.  xvi,  S.  268. 


Historical  sketch  on  Immunity  533 

obtained,  in  animals  well  immunised  against  the  cholera  vibrio, 
a  serum  endowed  with  a  high  anti-infective  power  but  entirely  with- 
out any  antitoxic  property.  The  guinea-pigs  themselves,  very  resistant 
to  the  cholera  peritonitis,  were  found,  on  the  other  hand,  to  be  very 
susceptible  to  the  minimum  lethal  dose  of  the  cholera  poison.  The 
absence  of  antitoxic  power  in  the  fluids  of  the  body  taken  in  con- 
nection with  a  well-marked  phagocytic  reaction  in  a  large  number 
of  cases  of  immunity,  natural  and  acquired,  has  turned  the  scale  in 
favour  of  the  cellular  theory.  The  impossibility  on  the  part  of  those 
who  maintain  the  purely  bactericidal  theory  of  the  body  fluids,  to  reply 
to  the  objections  above  mentioned  has  accentuated  this  favourable 
movement.  Just  at  this  moment,  when  the  theory  of  phagocytes  might 
be  regarded  to  have  obtained  the  rights  of  citizenship,  a  discovery  was 
made  which  appeared  to  overturn  it  completely.  I  have  mentioned 
more  than  once  that  the  attempts  of  the  partisans  of  the  bactericidal 
theory  of  the  body  fluids  have  failed  whenever  it  was  necessary  to 
give  evidence  of  their  action  in  the  refractory  animal.  Instead  of  a 
destruction  of  the  micro-organisms  in  these  fluids,  it  was  always  found 
that  they  perished  inside  the  phagocytes.  These  facts  have  even  led 
to  the  manifestation  of  a  desire  to  harmonise  the  humoral  theory 
with  the  theory  of  phagocytosis.  Denys,  with  certain  of  his  colla- 
borators, and  Buchner  and  his  pupils  came  to  the  conclusion  that  [5571 
the  alexins  are  merely  leucocytic  products.  As  regards  the  theory 
of  phagocytosis  we  have  this  section,  who  attribute  an  important 
function  in  healing  and  immunity  to  the  emigration  of  the  leucocytes 
towards,  and  their  accumulation  at  the  menaced  spot.  They  admit  • 
that  the  leucocytes  really  represent  the  healing  elements  of  the  animal 
body ;  it  is  not,  however,  they  say,  their  phagocytic  functions  which 
confer  upon  them  this  role  but  their  power  of  secreting  alexin.  These 
bactericidal  substances  act  outside  the  phagocytes— in  the  plasmas 
of  the  blood  and  of  the  exudations — and  phagocytosis  only  intervenes 
at  a  later  period  and  secondarily. 

This  new  modification  of  the  bactericidal  theory  of  the  body  fluids  j 
has  often  been  termed  by  Buchner  a  connecting  bridge  between  the  \ 
humoral  theory  and  the  cellular  theory  of  immunity. 

In  the  midst  of  this  movement  of  conciliation,  Pfeiffer1  in  1894 
published  a  work  on  the  immunity  of  the  guinea-pig  against 
experimental  cholera  peritonitis.  He  maintains  that  here  the 

1  Ztschr.  f.  Hyg.,  Leipzig,  1894,  Bd.  xvin  S.  1;  c£  also  Pfeiffer  u.  Issaeff,  ibid., 
1894,  Bd.  XVII,  S.  355. 


534  Chapter  XVI 

destruction  of  the  vibrios  takes  place  without  any  co-operation  on 
the  part  of  the  phagocytes  and  exclusively  by  means  of  the  body 
fluids.  The  vibrios,  before  their  complete  destruction  and  solution 
in  the  fluids  of  the  body,  are  transformed  into  granules,  presenting 
the  transformation  to  which  we  have  given  the  name  of  Pfeiffer's 
phenomenon. 

Several  of  Pfeiffer's  pupils  have  confirmed  his  view  in  connection 
with  the  cholera  vibrio,  and  have  extended  it  to  several  other  micro- 
organisms such  as  the  typhoid  coccobacillus.  The  destruction  of  the 
micro-organisms  in  these  cases  is  brought  about,  according  to  Pfeiffer 
and  his  collaborators,  not  by  the  alexins  of  Buchner,  but  by  a  separate 
substance.  The  protective  anti-infective  serum  contains  it  in  an 
inactive  state  only;  but  immediately  this  serum  is  introduced  into 
the  body  of  a  normal  animal,  the  bactericidal  substance  is  acted  upon 
by  the  endothelial  cells  and  becomes  "  active,"  capable  of  destroying 
a  large  number  of  vibrios.  Pfeiffer  has  developed  this  theory  more 
especially  in  an  article  published  in  1896,  entitled  "Ein  neues 
Grundgesetz  der  Immunitat1."  Pfeiffer's  observation  and  his  theory 
built  upon  it  gave  a  new  lease  of  life  to  the  humoral  theory  and  for 
some  time  many  observers  believed  that  the  theory  of  phagocytosis 
[558]  was  now  finally  overturned.  Frankel2  announced,  in  a  public  address, 
that  science  in  its  progressive  march  has  "discovered  the  methods 
of  defence  employed  by  the  animal  organism  against  its  most  dreaded 
enemies,  methods  which  have  nothing  in  common  with  phagocytosis, 
which  act  quite  independently  of  the  phagocytes  and  manifest  an 
action  so  energetic  that  we  may  calmly  eliminate  all  other  factors." 
This  view  is  based  on  the  discovery  of  antitoxins  and  the  bactericidal 
substance  studied  by  Pfeiffer. 

It  will  be  readily  understood  that  as  soon  as  I  learnt  of  the 
existence  of  a  real  extracellular  destruction  of  micro-organisms  I  at 
once  began  to  study  it  in  order  to  find  out  its  real  importance  amongst 
the  phenomena  of  immunity.  First  of  all,  I  examined  Pfeiffer's 
phenomenon  in  connection  with  the  cholera  vibrio3,  and  I  was  able 
to  show  that  it  was  produced  only  under  special  conditions.  The 
pre-existent  phagocytes  must  be  greatly  injured  before  the  cholera 
vibrios  can  be  transformed  into  granules.  Phagolysis  (so  I  termed 
this  transitory  damage  to  the  phagocytes)  is  indispensable  for  the 

1  Deutsdw  med.  Wchnschr.,  Berlin,  1896,  SS;  97,  119. 

2  "  Schutzimpfung  und  Impfschutz,"  Marburg,  1895. 

3  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  433. 


Historical  sketch  on  Immunity  535 

manifestation  of  Pfeiffer's  phenomenon  in  the  peritoneal  fluid.  When 
it  is  suppressed,  by  preparing  the  phagocytes  by  means  of  injections 
of  various  fluids,  we  find  that,  instead  of  Pfeiffer's  phenomenon, 
phagocytosis  is  almost  instantaneously  produced.  In  positions  where 
very  few  or  no  leucocytes  are  pre-existent,  as  in  the  subcutaneous 
tissue,  Pfeiffer's  phenomenon  is  never  observed. 

Even  in  the  case  of  the  cholera  vibrio  the  extracellular  destruction 
is  observed,  therefore,  only  in  special  cases.  Most  of  the  other 
pathogenic  micro-organisms  do  not  undergo  this  destructive  process 
at  all  under  conditions  in  which  the  cholera  vibrio  exhibits  Pfeiffer's 
phenomenon  in  a  marked  degree.  These  facts  appeared  to  justify  me 
in  the  conclusion  that  the  destruction  of  micro-organisms  takes  place 
in  the  animal  body  by  means  of  soluble  ferments,  the  result  of  phago- 
cytic  digestion.  These  ferments  are  found  under  the  normal  condition 
within  these  phagocytes  and  escape  from  them  when  they  are 
destroyed  or  receive  some  transient  injury.  This  conclusion  was  in 
flat  contradiction  to  the  theory  and  statements  of  Pfeiffer,  who 
attributed  an  important  function  to  the  endothelial  secretions.  To 
settle  this  controversy  I  tried  to  obtain  Pfeiffer's  phenomenon  outside 
the  body,  that  is  to  say  independently  of  any  co-operation  from  the  [559] 
peritoneal  endothelium.  It  is  sufficient  to  add  a  little  peritoneal 
lymph,  rich  in  leucocytes,  to  the  inactive  anti-infective  serum,  to 
obtain  in  hanging  drops  the  transformation  of  the  cholera  vibrios 
into  granules. 

Bordet1,  in  my  laboratory,  repeated  this  experiment  with  the 
object  of  determining  its  essential  mechanism.  He  succeeded  in 
obtaining  Pfeiffer's  phenomenon  in  vitro,  not  only  by  adding  peri- 
toneal lymph  from  a  normal  guinea-pig  to  the  specific  serum,  but 
also  by  adding  to  it  a  drop  of  fresh  blood  serum  from  the  same 
animal.  The  analysis  of  the  phenomena  which  take  place  under  these 
conditions  led  Bordet  to  the  following  hypothesis.  The  destruction 
of  micro-organisms  in  vaccinated  animals  takes  place  by  the  co- 
operation of  two  substances.  One  of  these  is  Buchner's  alexin  which 
is  found  normally  in  the  phagocytes ;  it  sets  up  bacteriolysis  properly 
so-called  when  it  is  enclosed  within  the  leucocytes  or  after  it  has 
escaped  from  them  at  the  time  of  phagolysis.  To  attain  this  end, 
however,  the  alexin  needs  the  co-operation  of  another  substance. 
This  is  the  protective  or  sensibilising  substance  of  Bordet.  It  circu- 
lates in  the  plasmas  and  carries  a  specific  character  which  is  absent 

1  Ann.  de  VInst.  Pasteur,  Paris,  1895,  t.  ix,  p.  462;  18f)G,  t.  x,  pp.  104,  193. 


536  Chapter  XVI 

from  the  alexin.  I  need  not  here  insist  at  any  length  on  this  theory, 
because  it  has  already  been  sufficiently  explained  during  the  course 
of  this  work. 

The  data  on  the  restricted  part  played  by  Pfeiffer's  phenomenon 
and  on  its  mechanism,  above  summarised,  have  been  attacked  by 
Pfeiffer  and  by  several  other  observers,  but  they  have  received 
general  confirmation,  so  that  their  accuracy  can  no  longer  be  in  doubt. 
Objections  were  also  raised  to  Bordet's  view  of  the  mechanism  of 
bacteriolysis.  Thus,  Abel  has  criticised  it  in  the  following  argument1 : 
"In  spite  of  the  soundness  and  the  boldness  of  the  majority  of  Bordet's 
statements  on  the  importance  of  the  various  factors,  and  especially 
of  the  leucocytes  in  immunity,  it  cannot  be  doubted  that  later 
researches  will  modify  and  correct  his  interpretations  which  we,  in 
Germany,  do  not  accept  in  their  full  extension.  Up  to  the  present, 
the  victory  in  the  various  rounds  has  always  been  with  Pfeiffer,  whose 
researches,  solid  and  exempt  from  bias,  have  made  him,  to  use  a 
sporting  expression,  the  'favourite'  with  all  those  who  follow  atten- 
[560]tively  the  international  contest  in  the  arena  of  the  problem  of 
immunity."  Abel  is  certainly  a  highly  esteemed  bacteriologist,  but 
he  is  not  a  good  prophet,  and  he  assumes  a  mistaken  attitude  in 
looking  at  the  subject  from  a  "  national "  point  of  view2.  In  Germany 
much  interest  is  taken  in  scientific  movements  and,  very  naturally, 
original  and  new  theories  are  there  criticised  and  discussed.  But 
that  does  not  justify  one  in  putting  forward  against  an  opinion  the 
statement  that  it  is  not  accepted  in  Germany.  In  this  country,  so 
rich  in  scientific  work,  we  find  partisans  of  the  most  opposite  views. 
In  any  case,  in  the  conflict  between  Pfeiffer  on  the  one  hand,  and 
Bordet  and  myself  on  the  other,  things  have  not  turned  out  as  Abel 
predicted.  The  two  substances  which  act  in  the  destruction  of  the 
micro-organisms  are  now  accepted  by  the  whole  world.  The  intimate 
relations  between  the  alexins  and  the  leucocytes  are  equally  recognised 

1  Centralblf.  Bakteriol  u.  Parasitenk.,  Jena,  1896,  Ite  Abt.,  Bd.  xx,  S.  766. 

2  It  would  clearly  be  wrong  to  take  one's  stand,  in  a  purely  scientific  question, 
on  a  national  point  of  view.    But  it  is  a  still  greater  mistake  to  look  at  matters,  in  the 
investigation  of  problems  which  concern  science  only,  from  a  personal  point  of  view. 
This,  however,  is  what  has  happened  several  times  in  the  discussion  of  phagocytosis. 
Certain  discontented  students  have  attempted  to  avenge  themselves  by  publishing 
works  and  criticisms  directed  against  the  theory  of  phagocytosis.     Having  no  doubt 
as  to  the  motive  for  these  publications  I  consider  myself  fully  justified  in  not 
referring  to  them  in  this  book,  in  which  I  have  taken  an  exclusively  scientific  point 
of  view,  and  in  which  I  have  endeavoured  to  weigh  as  carefully  as  possible  all 
criticisms  and  objections  that  have  been  directed  against  me. 


Historical  sketch  on  Immunity  537 

by  very  many  observers.  The  fact  that  the  alexins  are  confined 
within  phagocytes  has  been  confirmed  by  several  observers,  and  has 
received  a  very  convincing  proof  from  Gengou's  experiments  on  the 
comparative  action  of  the  serum  and  blood  plasma  against  micro- 
organisms. The  existence  of  phagolysis,  denied  at  first  by  some 
observers,  has  been  verified  by  others  and  can  now  no  longer  be 
doubted. 

The  relations  between  the  sensibilising  substance  and  the  phago- 
cytes are  less  easily  grasped  than  are  those  between  the  alexins  and 
the  leucocytes.  Nevertheless,  the  experiments  made  by  Pfeiffer  and 
Marx1,  have  led  these  observers  to  recognise  that  the  former  arises 
from  the  spleen,  the  lymphatic  glands,  and  the  bone  marrow,  that 
is  to  say,  organs  which  are  pre-eminently  phagocytic.  This  result 
has  been  confirmed  by  Deutsch  and  must  be  regarded  as  definitely 
settled.  All  the  data  collected  in  recent  years  have,  therefore, 
confirmed  the  view  that  the  destruction  of  micro-organisms  in 
the  refractory  animal  presents  itself  as  a  special  example  of  their 
absorption  by  formed  elements.  This  truth  was  so  fully  recognised  [SGI] 
in  our  laboratory  that  the  analogy  between  bacteriolysis  and  the 
destruction  of  animal  cells  was  looked  upon  as  quite  natural  and 
evident  Bordet  had  for  some  years  past  observed  that  the  blood 
serum  of  certain  animals  presented  a  marked  analogy  in  its  aggluti- 
native property  in  regard  to  micro-organisms  and  in  that  against 
red  blood  corpuscles.  In  1898,  studying  the  fate  of  the  spirilla  of 
the  goose  in  the  peritoneal  cavity  of  guinea-pigs  (see  Chapter  vi), 
I  observed  that  these  micro-organisms  underwent  the  same  changes 
both  within  and  outside  the  phagocyte;  this  fact  appeared  to  me  to 
be  in  perfect  harmony  with  the  whole  of  our  knowledge  concerning 
the  absorption  of  formed  elements  and  on  intracellular  digestion. 

Bordet2,  prepared  by  his  preceding  researches  on  the  agglutination 
of  the  red  blood  corpuscles,  set  himself  to  study  the  fate  of  the  red 
corpuscles  in  the  animal  body.  He  easily  established  a  close  relation- 
ship between  the  development  of  the  bacteriolytic  property  and  the 
haemolytic  power  of  the  serum  of  animals  prepared  by  repeated 
injections  of  bacteria  and  of  blood.  His  results  were  soon  (January, 
1899)  confirmed  by  Ehrlich  and  Morgenroth3,  who  supplemented  them 
with  the  important  statement  that  Bordet's  sensibilising  substance,  or 

1  Ztschr.f.  Hyg.,  Leipzig,  1898,  Bd.  xxvn,  S.  272. 

;  Ann.  de  I'lnst.  Pasteur,  Paris,  1898,  t.  xn,  p.  688 ;  1899,  t.  xin,  p.  273. 

3  Berl  klin.  Wchnschr.,  1S99,  S.  6. 


538  Chapter  XVI 

intermediary  substance  (E.  and  M.),  has  the  property  of  attaching  or 
fixing  itself  to  the  red  blood  corpuscles. 

The  works  on  haemolysis,  carried  out  during  the  last  three  years 
by  Ehrlich  and  Morgenroth  on  the  one  hand,  and  by  Bordet  on  the 
other,  have  allowed  us  to  extend  our  study  of  the  mechanism  of  the 
action  of  the  two  substances  on  micro-organisms  and  on  animal  cells. 
Ehrlich  has  extended  his  ingenious  theory  of  antitoxins  to  the  bacterio- 
lytic  substances,  which  he  regards  as  side-chains  detached  from  the 
cells  and  capable  of  absorbing  the  toxins.  In  a  series  of  remarkable 
investigations,  most  of  them  carried  out  in  collaboration  with  Morgen- 
roth, Ehrlich  has  developed  his  theory  which  attempts  to  offer  an 
account  of  the  essential  mechanism  which  presides  over  the  destruction 
of  micro-organisms  and  over  the  neutralisation  of  their  poisons.  This 
theory  is  at  present  in  full  swing  of  development.  Some  of  his  points 
contradict  several  of  the  conclusions  in  Bordet's  works.  Whilst  the 
latter  maintains  that  the  sensibilising  substance  becomes  fixed  as 
[562]  a  mordant,  Ehrlich  regards  it  as  entering  into  chemical  combination 
with  the  molecular  group  of  the  micro-organisms  and  of  the  animal 
cells.  According  to  Bordet,  the  alexin  of  the  same  species  of 
animal  is  always  the  same  substance.  Ehrlich  energetically  main- 
tains the  plurality  of  the  alexins,  to  which  he  gives  the  name  of 
complements. 

This  controversy  has  caused  a  most  interesting  exchange  of  views 
and  has  led  to  experiments  which  are  remarkably  ingenious ;  but  it 
must  be  admitted  that  as  yet  all  the  points  in  dispute  are  not 
definitely  settled.  It  is  evident  that  we  have  here  a  new  line  of 
research  which  promises  most  fruitful  results  for  science. 

We  have  described  in  various  chapters  of  this  work  the  funda- 
mental elements  of  Ehrlich's  theory.  Many  think  that  this  theory  is, 
in  principle,  antagonistic  to  the  theory  of  phagocytosis,  but  we  have 
already  observed  that  this  view  cannot  be  accepted.  It  is  true  that 
Ehrlich  maintains  that  the  bacteriolytic  and  cytotoxic  ferments 
which  we  have  called  cytases  (alexins  or  complements)  circulate  in 
a  state  of  solution  in  the  blood  plasma,  whilst,  according  to  the 
theory  of  phagocytosis,  they  are  found  under  normal  conditions 
inside  phagocytes.  But  this  view  has  nothing  to  do  with  the  basis 
of  the  theory  of  receptors,  or  of  Ehrlich's  side-chain  theory,  according 
to  which  the  antitoxin  and  certain  other  antibodies  (intermediary 
substance)  are  regarded  as  products  detached  from  cells  having  an 
affinity  for  the  toxins  and  the  microbial  products. 


Historical  sketch  on  Immunity  539 

The  theory  of  phagocytosis  seeks  to  establish  the  part  played  by 
these  cells  in  the  destruction  of  micro-organisms.  It  maintains  that 
the  vital  manifestation  of  the  phagocytes,  irritability,  mobility,  and 
voracity,  constitutes  an  essential  factor  in  ridding  the  animal  of 
micro-organisms,  because  the  true  bactericidal  ferment  is  contained 
within  the  phagocytes,  except  in  cases  of  phagolysis.  The  destruction 
of  the  micro-organisms  follows  the  laws  which  govern  the  absorption 
of  formed  elements  in  general.  This  absorption,  finally,  is  the  work 
of  two  soluble  digestive  ferments,  one  of  which  (fixative)  is  readily 
excreted  by  the  phagocyte  into  the  plasmas  of  the  blood  and  exuda- 
tions. The  theory  of  phagocytosis  seeks  to  establish  these  principles 
with  the  greatest  possible  exactness,  but  it  has  not  yet  ventured  to 
penetrate  more  deeply  into  the  phenomena  of  intracellular  digestion 
which  are  confounded  with  the  action  of  soluble  ferments  in  general. 
This  problem  is  still  far  from  being  satisfactorily  solved. 

In  spite  of  very  numerous  objections,  of  which  the  principal  ones  [563] 
have  already  been  mentioned,  the  theory  of  phagocytosis,  within  the 
limits  indicated,  so  far  from  being  overturned,  has  become  more  and 
more  consolidated,  thanks  to  the  numerous  observations  made  since 
its  foundation.  It  is  for  this  reason  that  the  opposition  has  calmed 
down  of  late  years  and  that  in  many  works  the  opinions  expressed 
have  become  more  favourable  to  the  role  of  phagocytosis  in  immunity. 

Soon  after  the  Congress  of  Hygiene  in  1891,  the  Pathological  Society 
of  London  devoted  several  meetings  to  a  discussion  of  the  question  of 
immunity.  Many  eminent  observers  took  part  in  these  debates,  which 
were,  in  general,  favourable  to  this  theory  of  phagocytosis1. 

At  the  International  Congress  of  Hygiene,  held  at  Budapest  in 
1894,  the  question  of  immunity  was  again  discussed.  Buchner2  made 
a  report  in  which  he  specially  insisted  on  the  leucocytic  origin  of  the 
alexins,  regarding  this  fact  as  particularly  capable  of  reconciling  the 
bactericidal  property  of  the  body  fluids  with  the  theory  of  phagocy- 
tosis. The  alexins,  however,  secreted  by  the  leucocytes,  must,  it  was 
assumed,  carry  out  their  principal  function  in  the  plasmas  of  the 
blood  and  exudations.  Phagocytosis  would  only  intervene  secondarily 
for  the  purpose  of  ingesting  the  micro-organisms  which  had  been 
already  killed  or  seriously  injured  by  the  alexins  of  the  body  fluids. 

1  Brit.  Med.  Journ.,  London,  1892,  VoL  i,  pp.  373,  492,  591,  604.  A  very  short 
summary  of  this  discussion  was  given  in  the  DeutscJw  med.  Wchnschr.,  Leipzig, 
1892,  S.  296. 

a  Munchen.  med.  Wchnschr.,  1894,  S.  717. 


540  Chapter  XVI 

In  his  last  summary  of  the  question,  presented  to  the  International 
Congress  of  Medicine  at  Paris  in  1900,  Buchner1  maintains  his  theory 
of  leucocytic  secretions.  But  he  already  takes  one  step  more  towards 
the  theory  of  phagocytosis,  at  least  as  regards  natural  immunity. 
He  consents  to  accept  the  fact  "that  phagocytic  activity  is  in  many 
cases  of  decisive  importance  in  overcoming  the  infective  processes, 
especially  in  those  cases  in  which  the  secreted  alexins  were  unable  to 
bring  about  more  than  a  temporary  attenuation  of  the  vital  functions 
of  the  bacteria.  Under  these  conditions  the  bacteria  could  only 
be  modified  in  so  far  as  their  chemical  functions  were  transformed 
into  a  latent  state,  from  which  they  would  be  ready  to  regain  their 
full  vital  activity  should  it  happen  that  the  phagocytes  were  not 
there  to  prevent  them  from  doing  so."  In  any  case  this  view  is 
[564]  widely  removed  from  the  old  theory,  according  to  which  phagocytes 
were  regarded  as  capable  of  ingesting  dead  and  inoffensive  bacteria 
only. 

A  second  adversary  of  the  theory  of  phagocytosis,  von  Behring2, 
gives  a  place  to  this  theory  not  only  in  certain  examples  of  natural 
immunity  but  even  in  some  cases  of  acquired  immunity,  e.g.  in  the 
immunity  of  sheep  vaccinated  against  anthrax,  an  example  I  have 
already  cited  in  Chapter  vm  (cf.  supra,  p.  242). 

It  would  take  too  long  to  describe  the  change  of  opinion  on  the 
theories  of  immunity  that  has  taken  place  during  recent  years.  I  will 
content  myself  with  citing  certain  examples  which  shall  be  taken  from 
the  works  of  declared  adversaries  of  the  theory  of  phagocytosis.  Thus, 
Fliigge,  who  early  declared  against  the  cellular  theory  completely  and 
categorically  and  at  the  same  time  argued  strongly  in  favour  of  the 
humoral  theory,  has  been  gradually  led  to  depart  from  his  first  position. 
We  may  follow  the  steps  of  his  conversion  in  the  different  editions 
of  his  Outlines  of  Hygiene.  In  the  first  edition  published  in  1889 
he  expresses  himself  in  the  following  manner3:  "Recent  researches 
indicate  the  probability,  however,  that  the  phagocytes  in  by  far  the 
greater  majority  of  cases  seize  the  infective  agents  which,  already 
dead,  are  not  in  a  condition  suitable  for  the  performance  of  a  defensive 
function.  On  the  other  hand,  it  is  proved  that  the  blood  and  blood 
plasma  of  warm-blooded  animals  possess  the  property  of  destroying, 
very  quickly,  enormous  numbers  of  pathogenic  bacteria," . . .  etc.  In 

1  Munchen.  med.  Wchnschr.,  1900,  S.  1193. 

a  Encydop.  Jahrbiicher,  Wien,  1900,  Bd.  ix,  S.  203. 

3  "Grundriss  der  Hygiene,"  Leipzig,  1889,  S.  487. 


Historical  sketch  on  Immunity  541 

the  fourth  edition  of  the  same  work,  published  in  1897,  we  find  at 
the  corresponding  place  the  following  passage1 :  "  Recent  researches 
indicate  the  probability,  however,  that  the  theory  of  Metschnikoff . . .  is 
not  in  a  position  to  offer  a  complete  explanation  of  the  process  of 
immunity."  This  passage  is  followed  by  a  somewhat  conciliatory  and 
eclectic  development  of  the  theory. 

Let  us  take  as  a  second  example  Giinther's  Introduction  to  the 
Study  of  Bacteriology,  widely  read  both  in  the  original  and  in  trans- 
lations. In  the  first  edition  published  in  18902  the  theory  of 
phagocytosis  is  curtly  dismissed  as  "  being  incapable  of  withstanding 
criticism."  In  the  fifth  edition  of  the  same  work,  however3,  published 
in  1898,  this  theory  is  no  longer  treated  thus  summarily.  It  is  given  [565] 
a  place  amongst  the  theories  of  immunity  and  an  attempt,  similar  to 
that  made  by  Buchner,  is  made  to  reconcile  it  with  the  humoral  theory. 

A  change  in  the  same  direction  may  also  be  observed  in  Charrin's 
view.  In  the  first  edition  of  his  Pathologic  gtntrale  infectieuse,  this 
observer4  had  already  taken  an  eclectic  view  on  this  question  of  the 
theories  of  immunity.  But  the  function  which  he  assigns  to  the 
phagocytes  is  subsidiary  and  secondary,  whilst  to  that  of  the  humoral 
properties  is  assigned  a  position  of  primary  importance.  In  the  second 
edition  of  the  same  work,  which  appeared  seven  years  later5,  the 
importance  of  phagocytosis  is  recognised  in  a  much  larger  measure, 
as  may  be  gathered  from  the  following  passages :  "  For  my  part, 
I  have  always  accepted  phagocytosis :  at  the  same  time  I  have 
always  accepted  the  existence  of  special  humoral  properties.  As 
early  as  1888  I  showed,  in  vivo,  that  the  germs  are  modified 
outside  the  cells  ;  but  I  did  not  know  from  what  groups  of  anato- 
mical elements  these  properties  were  derived,  I  exaggerated  their 
importance  and  it  is  the  decision  of  this  origin  and  this  importance 
that  renders  it  possible  to  reconcile  the  two  theories"  (p.  250). 
"After  all,  the  defence  rests  upon  these  two  great  processes  or 
cellular  activities,  phagocytosis  in  the  first  line,  and  then  humoral 
influences,  some  of  them  bactericidal  and  injurious  to  the  living  germ, 
others  antitoxic  and  injurious  to  their  secretions  "  (p.  253). 

Whilst  the  theory  of  phagocytosis  has  been  consolidated  by  the 

1  "  Grundriss  der  Hygiene,"  Leipzig,  4te  Aufl.,  1897,  S.  507. 

2  "Einfiihrung  in  das  Studium  der  Bakteriologie,"  Leipzig,  1890,  S.  146. 

3  "Einfiihrung  in  das  Studium  der  Bakteriologie,"  5te  Aufl.,  1898,  S.  275. 

4  "  Traite  de  medecine  "  de  Cbarcot,  Bouchard,  et  Brissaud,  1891, 1. 1,  pp.  219—230. 

5  "Traite  de  medecine...,"  2e  ed.,  1898,  t.  I,  pp.  250—254. 


542  Chapter  XVI 

demonstration  :  (1)  that  the  phagocytes,  in  cases  of  immunity,  ingest 
and  destroy  the  living  and  virulent  micro-organisms  without  the 
latter  needing  to  be  previously  deprived  of  their  toxins  ;  (2)  that  the 
phagocytes  absorb  toxic  substances  ;  (3)  that  the  phagocytes  contain 
bactericidal  cytases  and  produce  fixatives  ;  the  humoral  theories, 
in  spite  of  all  the  efforts  made  to  defend  them,  could  never  be 
developed  as  theories  that  were  in  the  slightest  degree  of  general 
application.  Certain  observers  who  from  the  first  were  very  sympa- 
thetic to  the  humoral  theories  have  attempted  to  give  a  complete 
summary  of  these  properties.  Thus,  Stern1  and  later  Frank2  have 
published  reports  drawn  up  with  great  care  and  in  a  very  impartial 
spirit  on  the  works  treating  of  the  properties  of  the  body  fluids 
[566]  and  the  part  they  play  in  immunity.  This  is  how  they  sum  up  the 
question.  Stern  came  to  the  conclusion  that  it  is  impossible  "to 
demonstrate  at  all  regularly  the  existence  of  relations  between  the 
bactericidal  action  of  the  blood  and  immunity  in  all  the  infective 
diseases.  In  some  cases,  however,  these  relations  are  so  marked 
that,  for  these  examples,  a  causal  bond  between  the  two  factors 
is  extremely  probable."  Frank  expresses  himself  in  the  following 
manner :  "  It  follows  most  clearly  that  the  immunity  of  an  animal- 
immunity  innate  or  acquired — corresponds  with  the  bactericidal 
property  of  the  blood  in  certain  exceptional  cases  only.  The.  only 
animal,  absolutely  susceptible  to  anthrax  and  whose  blood  is  entirely 
without  any  bactericidal  power,  that  it  is  at  present  possible  to 
cite,  is  the  mouse."  "The  bactericidal  action  of  the  blood  serum 
is  undoubtedly  a  fact  of  great  biological  importance  ;  but  equally 
certainly  it  cannot  be  the  general  cause  of  immunity,  whether  innate 
or  acquired." 

An  attempt  was  made  to  give  fresh  life  to  the  humoral  theory, 
either  by  assuming  that  the  bactericidal  substance  is  nothing  but 
the  eosinophile  or  pseudo-eosinophile  secretion  of  the  leucocytes 
(Kanthack),  or  by  supposing  that  for  the  destruction  of  micro- 
organisms in  the  animal  body  the  intervention  of  the  agglutinative 
substance  dissolved  and  distributed  in  the  body  fluids  is  essential 
(Max  Gruber).  These  two  views  were  put  forward  in  a  tentative 
form  and  as  preliminary  communications  only ;  there  is  no  possibility 

1  Centralbl.f.  allg.  Path.  u.  path.  Anat.,  Jena,  1894,  Bd.  v,  S.  212. 

2  Lubarsch  u.  Osier-tag's  "Ergebnisse  d.  allg.  Path.  u.  path.  Anat.,"  Wiesbaden, 
'  1895,  I.  Abt.,  S.  384. 


Historical  sketch  on  Immunity  543 

of  raising  them  to  the  dignity  of  theories,  and  of  late  years  they  have 
not  been  upheld. 

It  cannot  be  denied  that  not  one  of  the  humoral  theories  has  been 
able  to  retain  its  position  or  to  stand  against  the  numerous  facts  that 
have  been  accumulated  during  recent  years. 

This  extraordinary  discrepancy  between  the  bactericidal  power  of 
the  body  fluids  and  immunity  is  explained  by  the  circumstance  that 
the  microbicidal  substances  exist  in  the  living  animal  within  phagocytes 
and  only  escape  from  them  when  these  cells  have  been  injured.  The 
fact,  so  well  demonstrated  by  Gengou,  that  the  blood  plasma  is  without 
any  bactericidal  power  has  given  the  final  blow  to  the  microbicidal 
theory  of  the  body  fluids  and  it  can  no  longer  be  maintained. 

The  humoral  theories,  based  on  the  antitoxic  and  protective  power 
of  the  body  fluids,  can  claim  only  a  very  restricted  application.  These 
properties  are  met  with  in  acquired  immunity  only,  and  even  there 
are  not  constant.  Many  cases  of  acquired  immunity  against  micro-  [567] 
organisms  are  unaccompanied  by  any  antitoxic  power,  and  in  several 
examples  of  this  immunity  the  body  fluids  do  not  exhibit  any  pro- 
tective power. 

There  is  only  one  constant  element  in  immunity,  whether  innate  i 
or  acquired,  and  that  is  phagocytosis.  The  extension  and  importance  j 
of  this  factor  can  no  longer  be  denied. 

It  is  clearly  proved  that  phagocytes  are  susceptible  cells  which 
react  against  morbific  agents,  whether  organised  or  not.  These  cells 
ingest  micro-organisms  and  absorb  soluble  substances.  They  seize 
microbes  whilst  these  are  still  living  and  capable  of  exercising  their 
noxious  effect  and  bring  them  under  the  action  of  their  cellular 
contents,  which  are  capable  of  killing  and  digesting  the  micro- 
organisms or  of  inhibiting  their  pathogenic  action.  Phagocytes  act 
because  they  possess  vital  properties  and  a  faculty  of  exerting  a 
fermentative  action  on  morbific  agents.  The  mechanism  of  this  action 
is  not  yet  definitely  settled,  and  we  can  foresee  that  for  future 
researches  there  will  be  a  vast  and  fertile  field  to  be  reached  by 
pursuing  this  path. 

The  present  phase  of  the  question  of  immunity  constitutes  one 
stage  only  in  the  development  of  biological  science  and  one  which 
is  capable  of  many  improvements. 


[568]  CHAPTER  XVII 

SUMMARY 

Means  of  defence  of  the  animal  against  infective  agents. — Absorption  of  micro- 
organisms.— Phagocytes,  and  their  function  in  inflammation. — The  action  of 
phagocytes  in  the  absorption  of  micro-organisms. — The  cytases,  phagocytic 
ferments. — The  cytases  are  closely  bound  up  with  the  phagocytes. — The  fixatives 
and  their  function  in  acquired  immunity. — The  fixatives  are  excreted  by  the 
phagocytes  and  pass  readily  into  the  fluids  of  the  body. — Essential  mechanism 
of  the  action  of  the  fixatives. — Adaptation  of  phagocytes  to  destroy  micro- 
organisms in  acquired  immunity. — Difference  between  the  fixatives  and  the 
agglutinins.— Antitoxins  and  their  analogy  with  the  fixatives. — Hypotheses  as 
to  the  origin  of  antitoxins. — Cellular  immunity  is  a  fact  of  general  import. — 
Susceptibility  and  its  role  in  immunity. — Applications  of  the  theory  of  immunity 
to  medical  practice. 

\YHEN  an  animal  remains  unharmed  in  spite  of  the  penetration  of 
infective  agents  it  is  said  to  be  immune  to  the  diseases  usually  set 
up  by  these  agents.  This  idea  embraces  a  very  great  number  of 
phenomena  which  cannot  always  be  sharply  separated  from  allied 
phenomena.  On  the  one  hand,  immunity  is  closely  connected  with 
the  process  of  cure,  on  the  other,  it  is  related  to  the  disease.  An 
animal  may  be  regarded  as  unharmed  if  the  penetration  of  a  very 
dangerous  virus  sets  up  merely  an  insignificant  discomfort.  Never- 
theless, this  discomfort  is  accompanied  by  morbid  symptoms,  though 
they  may  be  very  slight.  It  is  useless  and  impossible  to  set  up  any 
precise  limits  between  immunity  and  allied  states. 

Immunity  presents  great  variability.  Sometimes  it  is  very  stable 
and  durable ;  in  other  cases  it  is  very  feeble  and  transient.  Immunity 
may  be  individual  or  it  may  be  generic.  It  may  be  the  privilege  of 
a  race,  of  a  species. 

Immunity  is  often  innate,  as  is  the  case  of  the  immunity  which  is 
called  natural.  But  it  may  also  be  acquired.  This  iast  category  of 
immunity  may  be  developed  either  by  natural  means,  after  an  attack 
of  an  infective  disease,  or  as  a  result  of  human  intervention.  The 


Summary  545 

principal  means  of  obtaining  artificial  acquired  immunity  consists  in 
the  inoculation  of  viruses  and  of  vaccines. 

Immunity  is  a  phenomenon  which  has  existed  on  this  globe  from  [569] 
time  immemorial.  Immunity  must  be  of  as  ancient  date  as  is  disease. 
The  most  simple  and  the  most  primitive  organisms  have  constantly 
to  struggle  for  their  existence  ;  they  give  chase  to  living  organisms 
in  order  to  obtain  food,  and  they  defend  themselves  against  other 
organisms  in  order  that  they  may  not  become  their  prey.  When  the 
aggressor  in  this  struggle  is  much  smaller  than  its  adversary  the 
result  is  that  the  former  introduces  itself  into  the  body  of  the  latter 
and  destroys  it  by  means  of  infection.  In  this  case  it  takes  up  its 
abode  in  its  adversary  in  order  to  absorb  the  contents  of  its  host 
and  to  produce  within  it  one  or  more  generations.  The  natural 
history  of  unicellular  organisms,  both  vegetable  and  animal,  often 
presents  to  us  these  examples  of  primitive  infection. 

But  infection  also  has  its  counter.  The  attacked  organism  defends 
itself  against  the  little  aggressor.  It  protects  itself  by  interposing  a 
resistant  membrane,  or  it  uses  all  the  means  at  its  disposal  to 
destroy  the  invader.  As  a  very  large  number  of  organisms,  in 
order  to  obtain  nourishment,  are  obliged  to  submit  their  food  to 
digestion  by  various  chemical  substances,  they  utilise  these  sub- 
stances in  the  struggle  against  the  infective  agents.  They  digest 
them  whenever  they  are  able  to  do  so. 

One  of  the  most  primitive  of  organisms,  the  plasmodium  of  the 
Myxomycetes,  which  is  composed  of  formless  protoplasmic  masses 
intermediate  between  lower  animals  and  plants,  ingests  foreign 
bodies  of  various  kinds.  It  often  happens  that  it  incorporates 
numerous  bacteria  which  are  growing  alongside  it  on  rotten  wood 
or  elsewhere.  The  plasmodium  allows  them  to  live  for  some  time 
within  its  digestive  vacuoles.  But  in  the  end  it  digests  them  by 
means  of  its  soluble  ferments,  substances  intermediate  between 
pepsin  and  trypsin.  Owing  to  this  digestive  power  the  plasmodia 
are  not  attacked  by  bacterial  infections. 

This  example,  taken  from  amongst  the  most  simple  organisms, 
may  serve  as  a  prototype  for  the  phenomena  of  immunity  in  general. 
At  the  commencement  of  the  study  of  this  remarkable  property  of  so 
many  living  organisms  it  was  thought  that  the  pathogenic  micro- 
organisms encountered,  Avithin  the  refractory  organism,  a  medium 
which  did  not  allow  them  to  live,  either  because  of  the  absence 
of  certain  nutritive  substances  indispensable  for  their  existence  or 
B.  35 


546  Chapter  XVII 

because  it  contained  some  substance  injurious  to  micro-organisms. 
[570]  Very  numerous  and  detailed  researches  have  demonstrated  the 
incorrectness  of  these  hypotheses.  There  are,  of  course,  certain 
pathogenic  micro-organisms  which  are  very  exacting  as  regards  the 
medium  in  which  they  will  grow.  Some  will  develop  only  in  the 
presence  of  particular  substances,  whilst  others  are  extremely  sensi- 
tive to  the  slightest  traces  of  poisons.  These,  however,  are  quite 
the  exception.  The  great  majority  of  pathogenic  micro-organisms 
belonging  to  the  group  of  bacteria  readily  adapt  themselves  to 
all  kinds  of  culture  media,  and  most  of  them  live  and  develop 
freely  in  the  blood  or  other  fluids  of  refractory  organisms.  This, 
therefore,  is  not  the  cause  of  the  immunity  in  such  organisms.  The 
cause  must  be  sought  for  amongst  factors  more  closely  connected 
with  life. 

Wishing  to  penetrate  more  deeply  into  these  phenomena  the 
hypothesis  was  put  forward  that  the  unharmed  organism  got  rid 
of  the  infective  micro-organisms  by  expelling  them  to  the  outside 
along  with  the  excreta.  It  was  maintained  for  a  considerable  time 
that  the  animal  organism  possessed  the  means  of  causing  pathogenic 
bacteria  to  pass  into  the  kidneys,  whence  they  were  eliminated  by 
the  urine.  It  had  to  be  acknowledged,  however,  that  this  elimination 
never  takes  place  in  cases  of  immunity,  and  only  comes  into  operation 
when  the  animal  is  ill  and  the  integrity  of  the  renal  filter  is  im- 
paired. 

The  infective  micro-organisms,  after  they  have  entered  into  the 
unharmed  organism,  remain  there  for  a  longer  or  shorter  period, 
and  perish  without  being  expelled.  This  disappearance  of  the 
micro-organisms  takes  place  by  the  same  mechanism  that  rids 
the  plasmodium  of  those  bacteria  which  it  has  managed  to  ingest 
during  its  slow  peregrinations  over  dead  leaves  or  rotten  wood. 
The  micro-organisms  are  absorbed  into  the  refractory  organisms 
as  the  result  of  a  true  act  of  digestion.  It  is  very  remarkable  that 
the  gastro-intestinal  ingestion,  so  well  provided  with  means  of  render- 
ing the  most  varied  aliments  soluble,  is  generally  incapable  of 
digesting  pathogenic  or  other  micro-organisms.  It  is  very  rare  to 
meet  with  soluble  ferments  of  the  intestinal  canal  which  are  capable 
of  digesting  microscopic  organisms,  especially  bacteria.  Consequently 
this  organ,  so  rich  in  digestive  diastases,  is  generally  inhabited  by 
a  large  number  of  bacteria  and  other  micro-organisms. 

Even  in  animals  whose  food  contains  large  numbers  of  micro- 


Summary  547 

organisms,  e.g.  the  larvae  of  flies,  the  digestive  juices  are  powerless 
to  destroy  them.  Nevertheless,  there  are  organisms  which  feed 
exclusively,  or  almost  exclusively,  on  bacteria  and  which  are  quite 
capable  of  digesting  them.  These  are  the  Protozoa,  such  as  the 
Amoebae  and  certain  Infusoria,  which,  without  any  trace  of  a  [571] 
digestive  tube,  easily  bring  about  this  result.  Amoebae  can  be 
grown  on  the  surface  of  agar  by  taking  care  to  sow  along  with  them 
bacteria  for  their  nourishment.  It  is  only  necessary  to  give  them 
a  single  species  of  micro-organism,  and  this  may  be  selected  from 
the  pathogenic  forms,  such  as  the  cholera  vibrio  or  the  BaciUus  coli. 
The  Amoebae  ingest  a  number  of  these  bacteria  in  the  living  state. 
They  then  kill  them  and  digest  them  in  their  digestive  vacuoles 
which  contain,  along  with  a  little  acid,  a  ferment  belonging  to  the 
trypsin  group,  the  amoebodiastase. 

The  bodies  of  lower  and  higher  animals,  alike,  are  very  rich  in 
elements  which  closely  resemble  the  Amoebae.  Sometimes  these  are 
to  be  found  in  the  epithelial  cells  of  the  digestive  canal  which  put 
out  protoplasmic  processes  for  the  purpose  of  seizing  food  and  trans- 
ferring it  to  their  interior,  where  it  is  submitted  to  the  action  of 
digestive  ferments.  Sometimes  they  are  the  cells  disposed  between 
the  body  wall  and  that  of  the  intestinal  canal,  which  float  freely 
in  the  fluids  of  the  body  or  are  more  or  less  fixed  in  the  interstitial 
tissue.  The  animal  kingdom  presents  a  great  variety  of  these 
amoeboid  elements,  known  under  the  general  name  of  phagocytes 
(cells  capable  of  devouring  solid  bodies).  One  of  the  most  primitive 
arrangements  of  phagocytes  is  met  with  in  Ascaris  and  its  allies 
belonging  to  the  group  of  the  Xeniatoda.  All  the  organisation 
that  these  round  worms  possess  consists  merely  of  four,  or  a  few 
more,  enormous  cells  attached  to  the  body  wall.  These  are  phago- 
cytes which  push  out  processes  of  enormous  length,  capable  of 
exploring  the  whole  of  the  internal  cavity  of  the  body. 

The  majority  of  phagocytes  circulate  in  the  lymph  and  blood  and 
pass  into  the  exudations.  These  white  corpuscles  have  a  comparatively 
uniform  structure  in  the  Invertebrata  and  present  themselves  as  small 
cells  with  a  nucleus  and  a  protoplasm  capable  of  amoeboid  move- 
ments. In  the  Vertebrata  we  meet  with  two  great  categories  of  white 
corpuscles,  of  which  one  group  resembles  those  of  the  Invertebrata 
in  that  they  also  possess  a  single  large  nucleus  and  an  amoeboid  proto- 
plasm. These  are  the  macrophages  of  the  blood  and  of  the  lymph, 
and  are  intimately  connected  with  the  macrophages  of  such  organs  as 

85—2 


548  Chapter  XVII 

the  spleen,  lymphatic  glands,  and  bone  marrow.  Another  group  of 
white  corpuscles  in  the  Vertebrata  is  made  up  of  small  amoeboid 
cells  which  are  distinguished  by  having  a  nucleus  which,  although 
single,  is  divided  into  several  lobes.  These  are  the  microphages 
[572]  whose  chief  peculiarity,  the  multi-lobed  form  of  the  nucleus,  must 
be  regarded  as  an  adaptation  for  the  purpose  of  passing  as  rapidly  as 
possible  through  the  walls  of  capillaries  and  small  veins. 

The  diapedesis  of  the  white  corpuscles,  their  migration  through 
the  vessel  wall  into  the  cavities  and  tissues,  is  one  of  the  principal 
means  of  defence  possessed  by  an  animal.  As  soon  as  the  infective 
agents  have  penetrated  into  the  body,  a  whole  army  of  white  corpuscles 
proceed  towards  the  menaced  spot,  there  entering  into  a  struggle 
with  the  micro-organisms.  Aided  by  the  special  form  of  their  nucleus 
the  microphages  are  the  first  to  pass  through  the  walls  of  the  vessels. 
Each  of  the  several  small  lobes,  into  which  the  nucleus  and  its 
protoplasm  is  divided,  passes  readily  through  the  minute  orifices 
between  the  endothelial  cells  of  the  vessels.  The  macrophages 
follow  the  microphages  and  become  mixed  in  greater  or  less  numbers 
with  the  exudations.  But  it  is  not  micro-organisms  only  which  set 
up  this  inflammatory  reaction  accompanied  by  the  emigration  and 
the  accumulation  of  leucocytes.  The  introduction  of  inert  bodies 
and  of  aseptic  fluids  brings  about  the  same  result.  The  phagocytes 
are,  as  a  matter  of  fact,  endowed  with  a  special  susceptibility,  which 
enables  them  to  perceive  exceedingly  small  changes  in  the  chemical 
or  physical  composition  of  the  medium  that  surrounds  them. 

The  leucocytes,  having  arrived  at  the  spot  where  the  intruders 
are  found,  seize  them  after  the  manner  of  the  Amoebae  and  within 
their  bodies  subject  them  to  intracellular  digestion.  This  digestion 
takes  place  in  the  vacuoles  in  which  usually  is  a  weakly  acid  fluid 
which  contains  digestive  ferments;  of  these  a  very  considerable 
number  are  now  recognised. 

Just  as  the  Amoebae  and  the  Infusoria  make  a  choice  from  amongst 
the  small  organisms  that  surround  them,  so  the  leucocytes  choose 
bodies  which  are  best  suited  to  their  use.  The  macrophages  seize 
by  preference  animal  cells  such  as  the  blood  corpuscles,  the  sperma- 
tozoa, and  other  elements  which  are  derived  from  animals.  Among  the 
infective  micro-organisms  the  macrophages  have  a ,  predilection  for 
those  that  set  up  chronic  diseases  such  as  leprosy,  tuberculosis,  and 
actinomycosis  and  also  for  those  which  are  of  animal  nature.  Into 
this  last  category  come  the  amoeboid  parasities  of  malaria,  Texas 


Summary  549 

fever  and  the  Trypanosomata.  The  macrophages  can  also  ingest 
the  bacteria  of  acute  diseases,  but,  save  in  exceptional  cases,  their 
intervention  is  of  little  moment. 

The  microphages,  on  the  other  hand,  appear  to  play  their  part  [573] 
specially  in  acute  infections.  Their  intervention  against  animal  cells 
is  nil,  or  almost  so.  Thus  they  rarely  seize  the  red  corpuscles  of 
the  same  or  of  a  foreign  species  of  animal.  They  also  appear  to  be 
repelled  by  parasites  of  animal  origin  and  by  certain  bacteria  which 
set  up  chronic  diseases.  Whilst  the  macrophages  seize  the  bacilli 
of  leprosy  with  great  avidity,  the  microphages  ingest  them  only 
exceptionally. 

The  morphological  and  physiological  differences  between  the  two 
great  categories  of  mobile  phagocytes  (leucocytes),  correspond  to 
differences  in  the  composition  of  their  soluble  ferments.  Just  as  the 
Amoebae  digest  their  prey  by  means  of  their  amoebodiastase,  a 
soluble  ferment  of  the  group  of  trypsins,  so  the  white  corpuscles 
submit  the  foreign  bodies  ingested  by  them  to  the  action  of  what  are 
now  known  as  cytases.  These  cytases  (alexins  or  complements  of 
other  writers)  are  soluble  ferments  which  also  belong  to  the  trypsin 
group.  They  act  in  a  medium  which  is  feebly  acid,  neutral,  or  feebly 
alkaline,  and,  like  the  amoebodiastase,  they  are  distinguished  by  a 
great  sensitiveness  to  heat.  When  the  cytases  are  contained  in  fluids, 
a  temperature  of  55° — 56°  C.  destroys  them  rapidly  and  completely. 
When  they  are  found  in  organs  reduced  to  the  state  of  an  emulsion, 
their  sensitiveness  diminishes  and  it  is  necessary  to  raise  the  tempera- 
ture to  58° — 62°  C.  in  order  to  destroy  their  activity. 

Bordet  maintains  that  the  cytases  are  very  different  in  the  various 
species  of  animals,  but  that  in  the  same  species  only  one  cytase 
exists.  Ehrlich  and  Morgenroth,  on  the  other  hand,  hold  that  the 
same  serum  contains  several,  sometimes  many,  different  cytases. 
This  question  is  too  difficult  to  be  definitely  solved  at  present.  It 
appears  to  me  very  probable  that  there  exist,  in  the  same  species  of 
animal,  two  different  cytases.  One  of  these,  the  macrocytase  which 
is  found  in  the  lymphoid  organs  and  in  the  serum  of  the  blood,  acts 
more  particularly  on  animal  cells.  Thanks  to  this  substance  an 
extract  or  maceration  of  the  spleen,  omen  turn  or  lymphatic  glands 
dissolves  the  red  blood  corpuscles  more  or  less  readily ;  these  extracts 
and  macerations,  however,  are  incapable  of  destroying  bacteria. 
When  the  macrophages  seize  the  nucleated  blood  corpuscles  they 
digest  them  completely,  not  sparing  even  the  nucleus,  so  resistant 


550  Chapter  XVII 

[574]  to  attack,  but  when  the  same  phagocytes  ingest  such  micro-organisms 
as  are  most  easily  digested,  such  as  the  cholera  vibrio,  their  action  is 
feeble.  The  vibrios,  without  any  transformation  into  granules,  re- 
main alive  for  some  time  and  are  destroyed  and  digested  with  very 
great  difficulty.  The  cytase  of  the  microphages,  or  microcytase,  is 
distinguished  by  other  properties.  It  destroys  and  digests  easily 
many  micro-organisms,  but  has  little  or  no  action  upon  the  red  blood 
corpuscles  and  other  animal  cells.  The  exudations  which  are  rich  in 
macrophages,  such  as  those  of  the  lymphoid  organs,  are  not  at  all 
or  only  slightly  bactericidal,  but  exhibit  a  solvent  action  on  red 
blood  corpuscles.  On  the  other  hand,  the  exudations,  which  are 
composed  in  great  part  of  microphages,  leave  red  blood  corpuscles 
intact,  but  readily  destroy  micro-organisms.  Similar  properties  dis- 
tinguish the  bone  marrow,  extracts  and  suspensions  of  which  do  not 
dissolve  red  corpuscles,  but  attack  micro-organisms.  Now,  we  know 
that  the  bone  marrow  is  the  principal  seat  of  origin  of  the  micro- 
phages. 

Even  after  the  addition  of  some  of  the  specific  fixative  to  the 
microphagic  exudations  no  solution  of  the  red  corpuscles  is  produced, 
which  demonstrates  most  clearly  that  the  microcytase  is  really  in- 
capable of  attacking  these  animal  cells. 

We  are,  therefore,  compelled  to  accept  the  existence  of  two 
different  cytases,  of  which  one  (the  macrocytase)  acts  specially  upon 
elements  of  animal  origin,  and  the  other  (the  microcytase)  acts 
principally  on  micro-organisms.  The  indication  of  any  more  de- 
tailed differentiations  is  impossible  in  the  present  state  of  our 
knowledge. 

There  are  certain  ferments  which,  during  the  life  of  the  cells 
which  produce  them,  pass  readily  into  the  surrounding  fluids.  For 
instance,  sucrase  can  be  recovered  without  difficulty  from  the  culture 
fluid  of  moulds  and  yeasts.  The  ferments  of  the  intestinal  digestion 
also  pass  with  great  facility  into  the  secreted  fluids.  Other  soluble 
ferments,  on  the  other  hand,  remain  very  closely  bound  up  with  the 
cells  which  manufacture  them.  Thus  the  zymase  of  the  yeasts  can 
only  be  freed  from  the  cells  of  these  fungi  with  great  difficulty,  under 
the  influence  of  great  pressure  and  under  conditions  which  pro- 
foundly alter  the  cell.  The  proteolytic  ferment  of  the  yeast  is  also 
very  adherent  to  the  cells  of  these  organisms.  The  fibrin-ferment,  or 
plasmase  of  the  white  corpuscles,  is  not  secreted  by  these  cells  so 
long  as  they  are  quite  intact.  But  it  is  sufficient  to  subject  them  to 


Summary  551 

unfavourable  conditions  of  existence  to  cause  them  to  throw  it  out 
from  their  bodies.     The  leucocytes,  when  removed  from  the  animal,  [575] 
undergo  a  deterioration  which  soon  leads  to  the  deposition  around 
them  of  filaments  of  fibrin. 

The  cytases  must  also  be  grouped  with  the  soluble  ferments  which 
are  not  thrown  off  by  the  phagocytes  so  long  as  these  remain  intact. 
Immediately  these  cells  are  injured,  however,  they  allow  a  part  of 
their  cytases  to  escape.  In  the  blood,  withdrawn  from  the  animal, 
the  white  corpuscles  allow  the  plasmase  to  pass  into  the  fluid,  where 
it  sets  up  the  coagulation  of  the  fibrin  and  the  formation  of  a  clot. 
At  the  same  time  these  cells  give  up  some  of  their  cytases  which 
communicate  to  the  serum  its  haemolytic  and  bactericidal  properties. 
This  fact  is  of  the  highest  importance  in  connection  with  the  question 
of  immunity.  The  best  demonstration  of  this  has  been  furnished 
by  a  comparison  of  the  bactericidal  power  in  the  different  parts  of 
the  body  and  in  the  body  fluids  extracted  from  the  animal. 

When  micro-organisms  are  introduced  into  those  situations  in  the 
refractory  animal  which  contain  pre-existent  leucocytes,  the  leuco- 
cytes, under  the  influence  of  the  shock,  undergo  serious  lesions, 
accompanied  by  the  throwing  out  of  the  cytases.  Under  these  con- 
ditions the  least  resistant  micro-organisms  (such  as  the  cholera  vibrio) 
exhibit  undeniable  signs  of  deterioration :  they  become  transformed 
into  granules  and  may  even  die  in  greater  or  less  numbers.  When, 
however,  the  leucocytes  are  well  protected  and  withstand  the  in- 
jection of  the  micro-organisms  without  being  profoundly  altered,  the 
extracellular  destruction  of  the  micro-organisms  does  not  take  place. 
On  the  contrary,  a  very  rapid  phagocytosis  is  produced  which  brings 
about  the  death  and  intracellular  digestion  of  these  micro-organisms. 
Under  these  conditions  vibrios  are  also  transformed  into  granules 
and  perish,  but  only  within  the  leucocytes.  The  phenomena  I  have 
just  mentioned  are  brought  about  in  the  peritoneal  cavity  and  in 
the  blood  vessels  of  refractory  animals,  that  is  to  say,  in  situations 
rich  in  leucocytes. 

In  the  subcutaneous  tissue,  in  the  fluids  of  oedemas  and  in  the 
anterior  chamber  of  the  eye  of  these  same  refractory  animals,  the 
phenomena  are  very  different.  As  in  these  situations  there  are  no 
pre-existing  leucocytes  or  their  number  is  insignificant,  the  micro- 
organisms introduced  do  not  suffer  serious  injury;  they  continue  to 
live  up  to  the  moment  when  the  leucocytes,  having  come  up  as  the 
result  of  the  inflammatory  reaction,  seize  them  alive,  kill  them, 


552  Chapter  XVII 

[576]  and  digest  them  within  their  substance.  Just  as  it  is  easy,  in 
situations  populated  by  pre-existing  leucocytes,  to  suppress  the 
extracellular  destruction  of  the  micro-organisms  by  preserving  the 
phagocytes  against  injury  or  phagolysis,  so  this  same  extracellular 
destruction  is  easily  set  up  in  situations  where  leucocytes  are  absent. 
When,  after  exudations  rich  in  leucocytes  have  been  injected  into 
the  subcutaneous  tissue,  we  introduce  micro-organisms  which  are  not 
very  resistant,  such  as  the  cholera  vibrio,  it  is  observed  that  these 
vibrios  are  destroyed  outside  the  cells,  having  first  been  transformed 
into  granules. 

There  can  be  no  doubt  as  to  the  conclusion  to  be  drawn  from  these 
various  experiments.  The  microcytase  is  the  substance  which  trans- 
forms the  vibrios  into  granules.  It  is  within  the  microphages,  when 
they  remain  intact,  that  the  vibrios  undergo  transformation.  When, 
on  the  other  hand,  the  microphages  are  injured  and  allow  the  micro- 
cytase to  escape,  the  transformation  of  the  vibrios  into  granules 
and  their  partial  destruction  take  place  in  the  plasmas  outside 
the  phagocytes. 

This  conclusion  is  supported  by  comparative  researches  on  the 
bactericidal  power  of  the  serum  and  of  the  blood  plasma  outside  the 
animal.  It  is  true  that  it  is  impossible  to  prepare  a  fluid  which 
shall  in  all  respects  be  comparable  to  the  plasma  of  the  circulating 
blood.  There  is,  however,  always  a  means  of  obtaining  outside  the 
animal  a  fluid  which  approaches  much  more  closely  to  blood  plasma 
than  does  serum.  Gengou  succeeded  in  preparing  in  tubes  coated 
internally  with  paraffin  a  fluid  which  coagulates  very  tardily,  and 
which  contains  very  little  fibrin-ferment.  This  fluid  is  found  to  be 
much  less  bactericidal  than  is  the  blood  serum  of  the  same  animal. 
It  is,  indeed,  often  found  to  be  entirely  without  bactericidal  power, 
whilst  the  corresponding  serum  is  capable  of  destroying  a  large 
number  of  micro-organisms. 

In  the  phenomena  of  the  absorption  of  cells  also  a  great  number 
of  facts  are  met  with  which  demonstrate  that  the  macrocytase 
escapes  from  the  macrophages  at  the  moment  of  their  phagolysis 
only.  For  example,  the  extracellular  solution  of  the  red  corpuscles 
takes  place  easily  in  the  peritoneal  fluid  of  animals  prepared  by 
a  previous  injection  of  the  same  corpuscles.  When  the  leucocytes 
of  the  peritoneal  cavity  are  abandoned  to  their  fate,  a  marked 
phagolysis  is  produced  and  consequently  a  solution  of  the  red 
corpuscles  in  the  fluid  itself.  When,  on  the  other  hand,  phagolysis 


Summary  553 

is  prevented,  the  macrophages  remaining  intact  do  not  allow  their  [577] 
macrocytase  to  escape  and  the  solution  of  the  red  corpuscles  takes 
place  almost  exclusively  inside  the  phagocytes. 

In  certain  animals  the  blood  serum  arrests  the  movements  of  their 
own  spermatozoa  at  once,  whilst  these  remain  quite  motile  in  the  animal 
itself.  This  is  due  to  the  fact  that  the  immobilising  macrocytase 
is  contained  within  the  macrophages  and  does  not  escape  from  them 
so  long  as  these  cells  remain  intact.  When,  in  such  animals,  their 
own  spermatozoa  are  introduced  into  the  subcutaneous  tissue,  they 
remain  motile  for  a  long  time;  when,  on  the  contrary,  the  sperma- 
tozoa are  injected  into  the  peritoneal  cavity,  where  the  leucocytes 
have  not  been  prepared,  phagolysis  is  produced  at  once  and  the 
spermatozoa  become  motionless  immediately. 

As  all  these  data  agree  in  demonstrating  that  the  uninjured 
phagocytes  retain  the  cytases — which  remain  within  them,  and  are 
not  found  in  the  surrounding  fluids, — we  can  readily  understand  the 
reason  for  the  differences  between  the  phenomena  of  immunity  and 
the  bactericidal  power  of  the  body  fluids.  The  rat's  serum  is  capable 
of  destroying  a  large  number  of  anthrax  bacilli,  although  these 
rodents  are  certainly  susceptible  to  anthrax.  The  reason  for  this 
is  that  in  the  serum  of  the  rat  the  bacilli  are  destroyed  by  the 
microcytase  which  is  set  at  liberty,  whilst  in  the  body  of  the  animal 
it  remains  enclosed  within  the  bodies  of  the  living  microphages. 
So  long  as  these  cells  exhibit  a  negative  chemiotaxis  against  the 
anthrax  bacillus,  the  micro-organism  remains  in  the  plasma,  where 
it  is  not  interfered  with.  Thanks  to  this,  multiplication  of  the  bacilli 
goes  on  in  the  body  of  the  animal,  the  micro-organism  killing  it  after 
becoming  generalised  in  the  blood  and  in  the  organs.  The  suscepti- 
bility of  the  leucocytes  is,  then,  the  cause  of  the  death  of  the  rats 
from  anthrax,  the  organism  of  these  rodents  being  unable  to  take 
advantage  of  its  richness  in  bactericidal  microcytase. 

Another  paradoxical  fact  is  met  with  in  guinea-pigs  immunised 
against  Gamaleia's  vibrio  (Vibrio  metchnikovi).  As  demonstrated  by 
von  Behring  and  Nissen,  the  blood  serum  of  these  guinea-pigs  is 
very  bactericidal  for  the  vibrio  in  question.  A  contact  of  less  than 
an  hour  is  quite  sufficient  to  destroy  large  numbers  of  the  micro- 
organisms. Nevertheless,  when  a  small  dose  of  a  culture  is  injected 
subcutaneously  into  these  hyper  vaccinated  guinea-pigs,  the  vibrios 
remain  alive  for  several  days,  up — indeed,  to  the  moment  when  they 
are  ingested  and  destroyed  by  the  leucocytes  which  come  up  in  large 


554  Chapter  XVII 

numbers  to  the  menaced  spot.  This  apparent  contradiction  is  easily 
explained  by  the  fact  that  it  is  in  the  serum  only  that  the  vibrios  en- 
counter the  microcytase,  which  has  escaped  from  the  microphages  at 
the  time  of  the  formation  of  the  clot  and  the  separation  of  the  serum. 
[578]  Alongside  those  cases  in  which  the  serum  of  susceptible  animals 
is  found  to  be  very  bactericidal,  examples  are  not  wanting  where 
the  blood  and  the  serum  of  refractory  animals  are  entirely  without 
this  power.  For  instance,  the  pigeon  is  refractory  to  Pfeiffer's 
influenza  bacillus,  but  the  blood  of  the  pigeon  forms  the  best  culture 
medium  for  this  micro-organism.  The  dog  is  refractory  to  the 
anthrax  bacillus,  against  which  the  blood  serum  of  the  same  animal 
is  not  at  all  bactericidal.  The  cause  of  this  absence  of  parallelism 
between  immunity  and  the  bactericidal  power  of  the  serums  must  be 
sought  in  the  difficulty  with  which  the  cytases  escape  from  the  leuco- 
cytes, and  also  in  the  modifications  which  they  may  undergo,  once  they 
are  distributed  in  the  fluids. 

In  cases  of  natural  immunity,  the  cytases  rid  the  animal  of  the 
micro-organisms  without  the  slightest  observable  co-operation  on  the 
part  of  other  soluble  ferments.  It  is  impossible  to  settle  definitely 
even  the  question  whether,  in  animals  which  enjoy  this  innate  im- 
munity, there  exists,  alongside  the  microcytase,  any  ferments  which 
come  to  its  aid.  The  conditions  are  quite  otherwise  in  a  very  large 
number  of  cases  of  acquired  immunity.  Here  it  is  found,  as  a  fairly 
general  rule,  that  in  addition  to  the  microcytases  there  exist  other 
substances  whose  r61e  in  the  defensive  action  offered  by  the  animal 
against  micro-organisms  is  very  important.  These  substances  are 
fixatives  which  co-operate  in  a  remarkable  fashion  with  the  bacteri- 
cidal action  of  the  cytases ;  but  whilst  these  latter  injure  the 
bacterial  cell  directly,  the  fixatives  do  not  interfere  with  its  life. 
The  bacteria,  permeated  by  fixatives,  may  even  continue  to  reproduce 
themselves  and,  under  certain  conditions,  to  invade  the  animal.  The 
fixatives,  then,  are  not  bactericidal,  but  by  fixing  themselves  upon 
the  micro-organisms  they  render  them  much  more  susceptible  to 
the  bactericidal  action  of  the  microcytases.  These  latter  are  further 
distinguished,  in  several  other  respects,  from  the  cytases.  The 
fixatives  must  also  be  classed  with  the  group  of  soluble  ferments, 
but  they  resist  much  higher  temperatures  than  those  which  destroy 
the  cytases.  Whilst  the  latter  are  quite  destroyed  at  55°  C.,  the 
fixatives,  to  be  completely  altered,  must  be  heated  to  beyond  60°  C. 
and  even  65°  C.  On  the  other  hand,  the  fixatives  are  distinguished 


Summary  555 

by  a  high  specificity  which  is  never  observed  in  the  cytases.  The 
majority  of  the  fixatives  are  incapable  of  fixing  themselves  upon 
more  than  a  single  species  of  bacteria  or  upon  a  single  class  of 
animal  cells,  and  only  certain  of  them  can  fix  themselves  upon  [579] 
allied  species  or  cells,  such  as  the  red  corpuscles  of  several  species 
of  animals.  In  these  cases,  too,  there  exists  a  sharp  quantitative 
difference  between  the  fixation  on  the  different  formed  elements. 
The  same  microcytases  are,  on  the  other  hand,  able  to  attack  all 
kinds  of  micro-organisms,  and  the  same  macrocytases  attack  all  kinds 
of  animal  cells. 

We  have  seen  that  the  cytases  correspond  to  the  zyraase  and  to 
the  proteolytic  diastase  of  the  yeasts  in  the  sense  that  all  these  soluble 
ferments  adhere  with  tenacity  to  the  cells  which  produce  them  and 
contain  them.  The  fixatives,  in  this  respect,  approach  sucrase 
(invertin) :  these  various  soluble  ferments  pass  readily  into  the  fluids 
which  bathe  the  cells  that  produce  them.  The  fixatives  are  found 
not  only  in  the  blood  serums,  prepared  outside  the  body,  but  also  in 
the  blood  plasma,  whence  they  pass  into  the  fluids  of  the  exudations 
and  transudations.  Whilst  no  cytases  are  found  in  the  subcutaneous 
tissue,  or  in  the  clear  fluids  of  oedemas  containing  no,  or  almost  no, 
cells,  fixatives  are  not  absent  from  these  various  situations  just 
indicated.  For  this  reason,  when  micro-organisms  are  introduced 
subcutaueously,  they  are  not  found  to  be  altered  by  the  cytases, 
but  it  is  easily  seen  that  they  are  permeated  with  fixatives.  The 
same  rule  applies  to  the  fixatives  of  the  animal  cells.  In  the 
example  we  have  cited,  the  spermatozoa,  in  an  animal  whose  serum 
renders  these  cells  motionless,  remain  quite  motile  in  the  epididymis 
and  below  the  skin.  From  this  fact  it  may  be  concluded  that  these 
situations  contain  no  free  macrocytase.  It  is  sufficient,  however,  to 
add  to  these  motile  spermatozoa  a  drop  of  normal  serum  contain- 
ing macrocytase  to  stop  their  movements  at  once,  the  fixative 
being  well  distributed  in  the  plasma  of  the  living  animal.  The 
spermatozoa,  then,  were  sensibilised  by  the  fixative  which  was  found 
in  both  the  epididymis  and  in  the  subcutaneous  tissue. 

The  cytases  are  soluble  ferments  which  are  essentially  intra- 
cellular:  the  fixatives  are,  on  the  other  hand,  soluble  ferments 
which  are  humoral.  These  fixatives,  however,  although  circulating 
in  the  plasmas,  are  undoubtedly  of  cellular  origin.  This  fact  was 
first  demonstrated  by  Pfeiffer  and  Marx,  who  found  the  specific 
fixative  of  cholera  vibrios  in  the  "  haematopoietic  organs,"  that  is  to 


556  Chapter  XVII 

say,  in  the  spleen,  lymphatic  glands,  and  bone  marrow,  at  a  period 
[580]  when  there  was,  as  yet,  none  in  the  blood.  This  fact  has  been 
extended  to  other  examples  of  fixatives  of  micro-organisms,  and  it 
cannot  be  questioned  that  the  phagocytes  produce  these  soluble 
ferments.  Under  the  influence  of  the  introduction  of  micro-organisms 
into  the  body,  a  phagocytic  reaction  is  produced  which  has,  as  a 
consequence,  the  digestion  of  these  micro-organisms  and  the  pro- 
duction of  corresponding  fixatives.  There  is  every  reason  to  believe 
that,  in  these  cases,  it  is  the  microphages  which,  seizing  and 
digesting  the  micro-organisms,  produce  the  fixatives. 

But  the  macrophages  are  also  capable  of  producing  these  ad- 
juvant ferments.  Even  in  normal  animals  the  macrophagic  organs, 
such  as  the  spleen,  and  especially  the  mesenteric  glands,  contain 
fixatives  which  help  in  the  solution  of  the  red  blood  corpuscles. 
Into  this  group  of  facts  we  must  also  place  the  production  by  the 
mesenteric  glands,  as  well  as  by  certain  other  lymphoid  organs, 
and  the  leucocytes  of  exudations  and  the  blood,  of  enterokynase, — 
the  soluble  ferment  which  aids  the  digestive  action  of  trypsin. 
This  enterokynase  is  also  a  species  of  fixative ;  it  permeates  the 
flakes  of  fibrin  and  renders  them  much  more  accessible  to  the  in- 
fluence of  the  trypsins. 

The  fact  that  the  enterokynase  of  the  intestinal  digestion  corre- 
sponds in  so  many  respects  to  the  fixatives  which  act  in  the 
absorption  of  formed  elements  in  general  and  of  micro-organisms 
in  particular,  furnishes  a  further  proof  that  the  destruction  of  micro- 
organisms in  the  animal  is  an  act  similar  to  true  digestion. 

Phagocytes,  those  elements  which  accomplish  the  absorption  of 
micro-organisms  and  of  animal  cells,  those  holders  of  digestive 
cytases,  are  also  the  manufacturers  of  fixatives.  Having  brought 
about  this  absorption,  the  phagocytes  set  to  work  to  elaborate  large 
quantities  of  fixatives,  although  they  are  unable  to  increase  the 
amount  of  cytases  in  any  marked  degree.  The  fixatives,  produced 
in  abundance,  can  be  excreted  outside  the  phagocytes  and  pass  into 
the  blood  plasma,  and,  with  it,  into  the  fluids  of  exudations  and 
transudations.  But  this  excretion  is  not  an  indispensable  act  for  the 
functioning  of  the  fixatives.  As  these  ferments  prepare  the  way 
for  the  digestive  action  of  the  cytases,  it  is  necessary  only  that  they 
should  be  able  to  fix  themselves  on  the  formed  elements  before  the 
latter.  It  is,  therefore,  easy  to  explain  cases  of  acquired  immunity 
in  which  no  fixatives  are  found  in  the  body  fluids.  Such  examples 


Summary  557 

are  not  rare,  and  are  characterised  by  the  absence  of  any  protective 
action  on  the  part  of  the  blood  serum.  In  these  cases,  the  fixatives,  [58 1] 
whose  existence  is  very  probable,  remain  lodged  within  the  phago- 
cytes, just  as  are  the  cytases.  Within  these  digestive  cells  the 
fixatives  may  quite  well  fulfil  their  preparatory  role,  this  being 
followed  immediately  by  the  action  of  the  cytase.  The  same  rule 
may  apply  also  to  the  cases  of  absorption  in  the  unprepared  animal, 
where  fixatives  are  not  found  in  the  blood  serum,  but  where  they  are 
able  to  act  within  phagocytes. 

The  excretion  of  fixatives  into  the  plasmas,  which  constitutes  the 
rule  in  cases  of  acquired  immunity,  presents  an  analogy  with  the 
excretion  of  pepsin  into  the  blood.  This  soluble  ferment  can  and 
does  pass  habitually  from  the  stomach  into  the  blood  and  thence 
into  the  urine,  where  it  is  often  met  with.  As  the  pepsin,  which  only 
acts  in  an  acid  medium,  cannot  be  utilised  in  the  alkaline  blood 
plasma,  it  is  evident  that  its  excretion  is  only  the  consequence  of  a 
too  abundant  over-production. 

In  recent  years  great  attention  has  been  paid  to  the  essential 
mechanism  of  the  action  of  fixatives  on  the  formed  elements  on  the 
one  hand,  and  on  the  cytases  on  the  other.  According  to  Ehrlich, 
the  fixatives  are  bodies  intermediate  between  the  two.  In  pos- 
session of  two  haptophore  molecular  groups,  they  are  capable  of 
entering  into  chemical  combination  with  the  micro-organisms  or 
the  animal  cells  on  the  one  hand,  and  with  the  cytases  on  the  other. 
It  is  for  this  reason  that  Ehrlich  applies  to  them  the  name  of 
"  amboceptors  "  or  "  intermediary  substances."  Based  on  analogous 
examples  in  organic  chemistry,  Ehrlich  thinks  that  the  fixatives 
serve  to  introduce  the  cytases  into  the  cells  upon  which  they  have 
to  act.  Bordet  does  not  share  this  view  and  maintains  that  the 
action  of  the  fixatives  is  not  a  chemical  action  in  the  proper  sense 
of  the  word,  but  is  a  kind  of  mordanting  which  sensibilises  the  formed 
elements  to  the  fermentative  action  of  the  cytases.  According  to  him, 
the  fixatives  have  no  affinity  for  the  cytases  and  in  no  way  serve 
them  as  intermediaries,  for  which  reason  he  gives  to  them  the 
name  of  sensibilising  substances.  The  question  is  still  under 
discussion,  but  we  may  hope  that  it  will  soon  enter  into  its  final 
phase. 

According  to  Ehrlich's  theory,  the  fixatives  contain  no  product 
coming  from  the  micro-organisms  or  from  the  animal  cells  upon 
which  they  are  fixed.  The  fixatives  are,  according  to  him,  side- 


558  Chapter  XVII 

chains  or  receptors,  produced  in  excess  and  expelled  into  the  blood 
[582]  plasma  by  the  cells  which  produce  them.  Ehrlich  does  not  tell  us  to 
what  category  these  cells  belong  ;  he  maintains  only  that  these  cells 
must  be  in  possession  of  receptors  endowed  with  a  specific  affinity 
for  certain  molecular  groups  of  micro-organisms  and  of  animal  cells. 
As  soon  as  the  receptors  are  saturated  by  these  molecular  groups, 
the  cells  which  make  use  of  the  former  for  their  nutrition  produce 
them  in  superabundant  quantity.  The  cells  of  animals,  treated  with 
micro-organisms  and  their  soluble  products,  or  with  red  blood 
corpuscles  or  any  other  kind  of  element  of  animal  origin,  acquire 
the  property  of  elaborating  more  and  more  of  the  corresponding 
receptors,  a  large  proportion  of  which  are  expelled  into  the  blood 
plasma. 

The  common  point  between  Ehrlich's  theory  and  the  view  main- 
tained in  this  work  consists  in  the  admission  of  a  cellular  property 
which  develops  more  and  more  in  proportion  to  the  treatment  of  the 
animal  by  formed  elements  of  all  kinds.  As,  in  acquired  immunity 
against  micro-organisms,  the  fixatives  are  most  frequently  found  in 
the  body  fluids,  it  must  be  concluded  that,  in  all  these  cases,  the 
cells  which  produce  them  have  become  adapted  by  a  kind  of 
education  to  manufacture  increasing  quantities  of  fixatives.  But 
even  in  those  examples  of  acquired  immunity  where  fixatives  are 
not  found  in  the  plasmas,  we  must  accept  a  modification  of  the  cells 
which  resist  the  invasion  of  micro-organisms.  These  changes  in  the 
cellular  properties  constitute,  therefore,  the  most  general,  and  conse- 
quently the  most  important,  element  in  acquired  immunity  against 
micro-organisms. 

As  already  mentioned  Ehrlich  does  not  assign  any  position  to 
the  cells  which  exhibit  these  modifications.  It  must,  however,  be 
accepted  that  they  belong  to  the  category  of  phagocytes.  Indeed, 
the  phagocytes  put  themselves  into  most  intimate  contact  with  the 
micro-organisms  and  foreign  animal  cells,  and  it  is  in  the  phagocytic 
organs  that  the  fixatives  are  found  before  they  are  met  with  in 
the  blood  plasma.  It  may  then  be  concluded  that,  in  acquired 
immunity  against  micro-organisms,  the  phagocytes  become  adapted 
to  elaborate  the  fixatives  in  large  quantities,  of  which  a  portion  is 
excreted  into  the  body  fluids,  as  has  been  shown  in  many  examples  of 
such  immunity. 

The  progressive  adaptation  of  the  phagocytes  in  intracellular 
digestion  can  be  demonstrated  by  the  fact  that  in  an  immunised 


Summary  559 

animal  the  fixatives  are  found  more  especially  in  the  phagocytic 
organs.  The  leucocytes  which  digest  gelatine  exhibit  in  an  even  [583] 
more  distinct  fashion  the  modification  of  these  cells  in  animals  which 
have  received  several  injections  of  gelatine.  The  leucocytes  of  exuda- 
tions, when  the  fluid  is  removed,  become  much  more  fitted  to  digest 
the  gelatine  than  they  were  at  first. 

A  similar  adaptation  is  also  observed  in  intestinal  digestion,  which 
may  serve  as  a  fresh  point  of  comparison  between  the  iutracellular 
digestion  of  the  phagocytes  and  the  extracellular  digestion  in  the 
intestines.  The  pancreas,  in  order  to  secrete  its  soluble  ferments,  adapts 
itself  to  the  nature  of  the  food  which  passes  into  the  digestive  canal. 

The  fixatives  are  not  the  only  soluble  ferments  which  appear  in 
large  quantities  in  the  fluids  of  the  immunised  animal.  Very  often 
there  are  found  along  with  them  substances  which  agglutinate  the 
micro-organisms  in  animals  which  have  received  several  injections  of 
micro-organisms  of  the  same  or  an  allied  species.  The  same  fact 
is  observed  in  animals  treated  with  animal  cells.  Thus  the  fluids 
of  animals  injected  with  blood  corpuscles  become  agglutinative  for 
these  corpuscles. 

The  analogy  between  the  agglutinins  and  the  fixatives  is  so  great 
that  for  some  time  several  observers  assumed  them  to  be  one  and 
the  same  substance.  This  can  no  longer  be  upheld,  for  it  is  clearly 
demonstrated  that  the  property  of  the  body  fluids  to  agglutinate 
micro-organisms  and  animal  cells  is  different  from  that  which  brings 
about  their  permeation  by  fixatives.  The  agglutinins  resist  the 
same  temperatures  as  the  fixatives  ;  both  are  specific  to  the  same 
degree  and  pass  equally  from  the  cells  which  produce  them  into  the 
plasmas  of  the  blood,  lymph,  exudations,  and  transudations.  The 
agglutinins  capable  of  clumping  the  formed  elements  into  masses 
may,  under  certain  conditions,  render  their  ingestion  by  the  phago- 
cytes more  easy.  In  general,  however,  the  part  played  by  the 
agglutinins  in  acquired  immunity  must  be  regarded  as  of  little 
importance,  and  for  that  reason  we  abstain  from  basing  any  theory 
of  this  immunity  on  the  agglutinative  property  of  the  body  fluids. 
Besides  fixatives  and  agglutinins,  the  fluids  of  an  animal  which  has 
acquired  immunity  very  probably  possess  other  properties  which 
must  have  a  greater  or  less  function  in  acquired  immunity.  Thus, 
we  are  often  struck  by  the  stimulating  action  of  these  fluids  on  the 
normal  animal  into  which  they  are  introduced.  This  stimulation  is  [584] 
especially  manifested  against  the  phagocytic  reaction. 


560  Chapter  XVII 

As,  in  the  majority  of  cases  of  acquired  immunity,  the  blood 
serum  contains  fixatives  in  considerable  proportion,  and  as  these 
fixatives  aid  the  action  of  the  cytases  in  a  remarkable  fashion,  we 
can  readily  understand  that  the  introduction  of  such  a  blood  serum 
into  a  normal  animal,  unprepared  by  any  vaccination,  may  bring 
about  a  great  resistance  against  the  corresponding  pathogenic  micro- 
organisms. The  fixatives,  injected  with  the  serum,  fix  themselves 
with  avidity  upon  the  micro-organisms.  These  organisms  may 
become  a  more  ready  prey  to  the  phagocytes  and  be  destroyed 
very  rapidly.  In  particular  cases,  where  the  injection  of  microbial 
cultures  sets  up  a  phagolysis,  enough  cytases  are  thrown  out  to 
affect  the  microbes  already  sensibilised  by  the  fixative.  This  is 
followed  by  a  refractory  condition  of  the  animal  proportionate,  in 
general,  to  the  amount  of  fixative  serum  that  is  injected.  This 
kind  of  acquired  immunity,  conferred  by  serums  or  certain  other 
body  fluids  rich  in  fixative  substances,  has  often  received  the  name 
of  passive  immunity.  This  term  is  only  justified  in  those  rare  cases 
where  the  introduced  serum  itself  contains  a  sufficient  amount  of 
cytases  to  destroy  all  the  micro-organisms.  Most  often  it  is  the 
normal  animal  which  has  to  furnish  this  bacteriolytic  ferment. 
Now,  as  in  phagolysis  the  quantity  given  off*  is  too  small,  it  is  to 
the  co-operation  of  the  holders  of  cytases,  that  is  to  say,  to  the 
phagocytes,  that  the  animal  must  have  recourse.  The  phagocytes, 
being  susceptible  cells,  their  co-operation  can  only  be  counted  upon 
in  cases  where  they  exhibit  a  sufficient  activity.  When  these 
elements  are  weakened  by  narcotics  or  by  any  other  cause,  they 
become  incapable  of  intervening  with  efficacy  and  the  animal  falls 
a  victim  to  the  pathogenic  micro-organisms,  in  spite  of  the  more  than 
sufficient  amount  of  fixatives  that  was  introduced. 

In  natural  or  acquired  immunity,  it  is  the  resistance  of  the  animal 
against  the  micro-organisms  which  plays  the  principal  part.  The 
introduction  of  toxins  ready  prepared  is  only  done  under  artificial 
conditions,  as  in  laboratory  experiments.  Hence  we  see  that,  under 
natural  conditions,  it  is  against  the  penetration  of  the  micro- 
organisms that  the  animal  must  be  protected.  So  soon  as  these 
producers  of  poisons  can  no  longer  maintain  themselves  in  the 
immunised  animal  their  toxic  secretions  do  not  come  into  play. 
It  is  for  this  reason  that  animals  vaccinated  against  pathogenic 
micro-organisms  do  not  suffer  from  intoxication,  although  they  are 
[585]  by  no  means  insusceptible  to  the  microbial  poisons.  It  is  a  fact 


Summary  561 

of  the  highest  importance  from  the  point  of  view  of  immunity  in 
general,  that  the  resistance  offered  to  micro-organisms  in  no  way 
implies  insusceptibility  to  their  poisons.  The  view  has  frequently 
been  expressed  that,  in  acquired  immunity  at  least,  the  animal  must 
first  acquire  immunity  against  the  microbial  toxins,  after  which  the 
micro-organisms,  deprived  of  their  principal  weapon,  descend  to 
the  rank  of  inoffensive  saprophytes.  Such  cases  may  be  found, 
but  it  is  none  the  less  true  that  immunity  against  micro-organisms 
may  be  acquired  independently  of  that  against  the  toxins,  and  that 
this  constitutes  the  general  rule. 

Immunity  is  much  more  readily  acquired  against  micro-organisms 
than  against  their  toxins.  Hence,  antimicrobial  vaccination  was 
accomplished  by  science  before  that  against  their  toxins.  In  the 
early  researches  on  this  subject  antitoxic  immunity  appeared  to 
be  very  difficult  of  attainment,  and  it  was  only  after  the  discovery 
made  by  von  Behring,  who  inaugurated  a  new  path  in  microbiology, 
that  better  results  were  obtained.  Von  Behring  not  only  suc- 
ceeded in  immunising  animals  against  some  of  the  principal  microbial 
toxins,  he  demonstrated  the  existence  of  specific  antitoxins  in  their 
body  fluids. 

This  very  unexpected  conception  of  antitoxins  at  once  took  root 
in  science,  for  it  has  been  possible,  thanks  especially  to  the  remark- 
able works  of  Ehrlich,  to  extend  it  to  toxins  of  non-microbial  origin. 
We  are  already  acquainted  with  a  certain  number  of  antitoxins 
which,  however,  are  not  comparable  in  number  to  the  other 
antibodies.  Amongst  these,  the  fixatives  have  many  points  of 
analogy  with  the  antitoxins.  Like  them,  they  are  resistant  to  heat : 
they  exhibit  also  a  fairly  marked  specificity,  and,  like  the  fixatives, 
they  are  distributed  in  the  plasmas. 

In  the  presence  of  so  many  points  of  similarity  with  the  fixatives, 
one  is  tempted  to  attribute  to  the  two  categories  of  antibodies  the 
same  origin.  The  elaboration  of  antitoxins  by  the  phagocytic 
elements,  accumulated  in  the  blood  and  disseminated  in  the  organs, 
appears,  in  fact,  to  be  very  probable.  Certain  facts  bearing  on 
the  absorption  of  various  toxins  by  the  leucocytes,  as  well  as  the 
distribution  of  antitoxins  in  the  animal  body,  speak  in  favour  of 
this  view.  On  the  other  hand,  the  impossibility  of  attributing  the 
elaboration  of  antitoxins  to  cells  attacked  by  the  corresponding 
toxins  is  quite  in  harmony  with  the  same  hypothesis.  This  hypo- 
thesis is  especially  supported  by  the  numerous  facts  which  prove  the  [586] 
B.  36 


562  Chapter  XVII 

readiness  with  which  the  leucocytes  react  against  all  kinds  of 
poisons,  microbial  or  other  toxins,  as  well  as  against  organic  and 
mineral  poisons,  such  as  the  alkaloids  and  the  arsenical  combina- 
tions. However,  in  spite  of  so  many  data  which  speak  in  favour 
of  the  phagocytic  origin  of  antitoxins,  it  has  been  impossible  to 
support  this  view  by  rigorous  facts  easy  of  interpretation,  such  as 
those  which  science  possesses  in  support  of  the  phagocytic  origin  of 
fixatives. 

The  antitoxins  have  acquired  a  very  great  importance  in  the 
artificial  cure  of  toxo-infective  diseases,  the  aim  in  these  cases  being 
to  paralyse  the  action  of  the  toxins  already  produced  by  the  micro- 
organisms and  absorbed  by  the  diseased  animal.  But  their  function 
is  less  in  the  protection  against  diseases  where  the  object  to  be 
obtained  is  a  reaction  against  the  micro-organisms  before  these 
are  able  to  inundate  the  animal  with  their  toxic  secretions.  It  is 
for  this  reason  that  the  immunity  against  toxins  must,  in  the  study  of 
immunity,  occupy  a  less  preponderant  place  than  does  the  immunity 
against  micro-organisms. 

As  the  micro-organisms  placed  in  the  refractory  animal  ultimately 
undergo  a  digestion  by  chemical  substances  elaborated  by  the  phago- 
cytes, so  also  the  toxins  undergo  a  chemical  modification  due  to  the 
presence  of  substances  in  the  production  of  which  the  living  elements 
of  the  animal  play  a  large  part.  The  direct  action  of  antitoxins  on  the 
toxins,  so  well  demonstrated,  especially  by  Ehrlich's  investigations, 
does  not,  however,  exclude  the  intervention  of  living  cells,  which, 
though  sometimes  not  very  manifest,  is  in  other  cases  very  marked. 

The  reaction  of  the  living  elements  against  the  microbial  toxins 
and  their  allies  leads  to  the  production,  and  even  the  over-production 
of  antitoxins.  According  to  Ehrlich,  these  elements  are  the  receptors, 
or  side-chains,  which,  to  a  certain  extent,  pre-exist  in  the  cells 
which  are  capable  of  elaborating  the  antitoxins.  On  entering  into 
combination  with  the  toxin  molecules,  the  side-chains,  which  are 
indispensable  for  the  nutrition  of  the  cells,  are  reproduced  in  very 
large  numbers.  After  having  saturated,  so  to  speak,  the  productive 
elements  of  the  antitoxin,  the  superfluous  side-chains  escape  from 
the  cell  and  pass  into  the  plasmas  of  the  body  fluids.  This  theory 
may  be  brought  into  harmony  with  the  other  theory,  which  maintains 
that  certain  elements  of  the  animal,  capable  of  acting  on  the  complex 
molecules  of  microbial  toxins  and  their  allies,  produce  special  soluble 
[587]  ferments,  which  digest  the  toxins  whose  introduction  frequently 


Summary  563 

excites  the  hypersecretion  of  the  ferments.  Here  we  have  some- 
thing similar  to  the  hypersecretion,  by  the  glands  of  the  stomach,  of 
pepsin,  a  part  of  which  passes  into  the  blood  in  order  to  escape  with 
the  urine. 

According  to  Ehrlich's  theory,  the  antitoxins  are  only  capable  of 
neutralising  the  injurious  action  of  toxins  when  the  former  are  found 
dissolved  in  the  body  fluids.  The  same  receptors  which  fix  the 
toxins  in  the  plasmas  and  thus  prevent  them  from  reaching  the 
susceptible  elements,  bring  about  an  opposite  result  when  they  are 
found  inside  the  cells.  In  this  latter  case,  the  receptors,  owing  to 
their  great  affinity  for  the  toxins,  attract  them  and  allow  them  to  pass 
into  the  cells,  in  this  way  aiding  the  dangerous  function  of  the  toxo- 
phore  group. 

This  is  an  ingenious  idea,  conceived  to  bring  into  harmony 
a  certain  number  of  observed  facts.  In  the  present  state  of  our 
knowledge  it  cannot  be  subjected  to  rigorous  experimental  test. 
Many  well-established  facts,  however,  are  not  in  complete  accord 
with  this  hypothesis.  According  to  it  the  antitoxic  immunity 
resides  exclusively  in  the  body  fluids  ;  the  living  cells,  instead  of 
acquiring  immunity,  become  more  and  more  susceptible.  Under 
these  conditions  it  is  difficult  to  conceive  of  an  immunity  against 
poisons  of  the  simplest  organisms  ;  nevertheless,  this  certainly  exists. 
A  plasmodium,  which  becomes  adapted  to  all  kinds  of  toxic  sub- 
stances, acquires  an  immunity  against  them,  and  this  is  due  to 
changes  taking  place  in  the  living  elements ;  it  is  not  the  result  of 
modifications  in  the  toxic  fluids  which  bathe  them.  This  biological 
adaptation  is  observed  in  the  case  of  physical  factors  which  may 
interfere  with  the  life  of  these  primitive  organisms. 

On  the  other  hand,  it  must  be  accepted  that  the  living  cells  of  a 
complicated  and  higher  organism  may  also  acquire  immunity  against 
toxins.  The  first  example  of  this  kind  was  shown  in  relation  to  the  red 
blood  corpuscles  of  mammals  vaccinated  against  the  toxic  serum  of 
the  eel.  Whilst  the  body  fluids  of  immunised  rabbits  become  anti- 
toxic, their  red  blood  corpuscles,  when  completely  freed  from  the 
serum,  in  certain  cases  resist  the  action  of  the  eel's  serum.  It 
must  be  admitted  that  in  this  example  we  have  an  acquired  immunity 
of  the  cells  similar  to  that  met  with  in  lower  organisms. 

A  second  example  of  the  immunity  of  the  red  corpuscles  was 
observed  by  Ehrlich  and  Morgenroth  in  goats  prepared  by  injections  [588] 
of  the  blood  of  other  individuals  of  the  same  species.    In  this  case, 

36—2 


564  Chapter  XVII 

according  to  these  writers,  no  co-operation  by  antitoxin  is  met  with. 
The  body  fluids  of  the  goats  do  not  become  capable  of  neutralising  the 
toxin  of  the  haemolytic  serum,  whilst  the  red  corpuscles  themselves 
acquire  an  immunity  against  this  toxin,  an  immunity  entirely  cellular. 
Ehrlich  attempted  to  penetrate  into  the  essential  mechanism  of  the 
resistance  of  the  red  blood  corpuscles  on  the  supposition  that  these 
corpuscles,  instead  of  reproducing  their  receptors,  as  when  there  is 
production  of  antitoxin,  get  rid  of  them  entirely.  Deprived  of 
receptors,  they  can  no  longer  be  affected  by  the  haemolytic  cytase 
which,  as  Ehrlich  maintains,  only  penetrates  into  the  red  corpuscles 
owing  to  the  affinity  of  the  intermediate  substance  (fixative)  for  the 
receptor.  This  hypothesis  of  the  mechanism  of  acquired  cellular 
immunity  scarcely  accords  with  the  hypothesis  of  the  special  function 
attributed  to  the  receptors  in  the  nutrition  of  the  living  elements. 

Cellular  immunity  can  be  most  easily  demonstrated  in  relation  to 
the  red  corpuscles  of  the  blood,  as  these  elements  are  very  numerous 
and  are  capable  of  being  isolated  and  freed  from  the  fluid  in  which 
they  are  bathed.  For  this  reason,  science  does  not  as  yet  possess 
sufficiently  exact  data  on  the  immunity  of  other  cells  in  higher 
animals.  Many  facts,  however,  indicate  that  such  immunity  does 
exist.  There  are,  indeed,  living  elements  which  only  acquire  immunity 
with  great  difficulty  and  very  slowly.  Such  are  the  nerve  cells, 
elements  which  are  specially  susceptible.  Von  Behring  has  strongly 
insisted  on  the  fact  that  in  animals  subjected  to  repeated  injections  of 
bacterial  toxins,  the  nerve  centres  not  only  do  not  become  accustomed 
to  their  injurious  action,  but  even  acquire  a  hypersusceptibility  which 
is  often  very  great.  The  observation  is  perfectly  accurate,  but  it  is  none 
the  less  true  that  this  period  of  exaggerated  susceptibility  is  followed 
by  another,  during  which  the  susceptibility  becomes  less  marked  and 
ends  by  giving  place  to  a  true  adaptation.  We  are,  therefore,  com- 
pelled to  accept  the  fact  that  even  the  nerve  cells  are  no  exception  to 
the  general  rule,  but  are  able  to  acquire  a  diminished  susceptibility  to 
a  poison. 

Several  facts  of  another  series  confirm  this  conclusion.    In  the 

study  of  the  action  of  the  nervous  system  one  frequently  has  occasion 

to  observe  instances  of  adaptation.     I    will  cite    as   an  example 

[589]  the  adaptation  of  animals  to  spinal  concussion  studied  by  Le'pine1. 

By  percussing  the  lumbar  region  of  rabbits  and  guinea-pigs  we  may 

induce  in  them  an  immediate  paraplegia.     This  is  transitory,  and 

1  Compt.  rend.  Soc.  de  biol.t  Paris,  1900,  p.  385. 


Summary  565 

lasts  at  most  for  a  few  hours.  The  phenomenon  may  be  reproduced 
several  times  in  the  same  animal.  "But,"  remarks  Lepine,  "when  these 
experiments  are  continued  for  several  days  or  several  weeks,  striking 
always  at  the  same  level,  we  soon  observe  that  the  resistance  of  the 
animals  to  the  blows  increases  very  rapidly,  and  that  excitations 
which,  in  normal  animals,  produce  paraplegias  of  several  hours' 
duration,  produce  no  effect  upon  those  which  have  been  under 
experiment  for  several  days."  We  have  in  this  example  a  real 
adaptation  of  the  spinal  region  when  subjected  to  concussion. 

Similar  facts  are  known  to  everyone  as  an  experience  of  daily  life. 
We  can  become  habituated  more  or  less  easily  to  all  kinds  of  violent 
sensations.  Light  and  very  intense  noises  which,  at  first,  excite 
exaggerated  reflex  actions  are  ultimately  perceived  without  setting  up 
the  least  movement.  Even  in  the  psychical  sphere  habit  dulls  painful 
feelings,  and  it  is  very  probable  that  a  whole  gamut  of  adaptation, 
starting  from  unicellular  organisms  which  accustom  themselves  to 
live  in  an  unsuitable  medium,  up  to  cultured  human  beings  who 
habituate  themselves  to  a  disbelief  in  human  justice,  will  be  found 
to  rest  upon  one  and  the  same  fundamental  property  of  living  matter. 

Regarded  from  this  point  of  view,  immunity  becomes  a  very 
general  phenomenon,  passing  far  beyond  the  resistance  offered  by  the 
animal  to  infective  diseases.  After  all  is  said  and  done,  it  invariably 
reduces  itself  to  that  cellular  susceptibility  [irritability]  which  governs 
so  many  of  the  vital  phenomena  in  plants  and  in  animals.  It  is  this 
susceptibility  which  impels  the  branch  towards  the  light  and  the  root 
towards  the  ground,  and  which  guides  the  spermatozoon  towards  the 
ovum.  From  the  very  commencement  of  embryonic  life  the  cells 
derived  from  the  segmentation  of  the  egg  exhibit  a  marked  suscepti- 
bility. Wilhelm  Roux1  observed  that  the  earliest  cells  of  the  frog 
embryo,  if  they  are  separated  by  artificial  intervention,  guided  by 
their  positive  chemiotaxis  again  come  together.  In  the  formation  of 
the  tissues  cellular  susceptibility  plays  an  important  undoubted  role. 
The  prolongations  of  the  nerve  cells  direct  themselves  towards  the 
organs  of  sense  or  towards  the  muscular  fibres,  according  to  their  [590] 
specific  susceptibility2.  The  mother-cells  of  the  capillary  vessels  are 
also  guided  by  susceptibility,  when  they  go  towards  a  new-formed 

1  "Ueber  die  Selbstordnung  der  Furchungszellen,"  in  Berichte  d.  naturwiss. 
Vereins  zu  Innsbruck,  1893,  Bd.  XXL 

2  Herbst,  Biol  Centralbl.,  Erlangen,  1894, 1895,  Bde.  xiv,  xv;  Forssmanii,  Zieglers 
Beitr.  z.path.  Anat.,  Jeiia,  1898,  Bd.  xxiv,  S.  56. 


566  Chapter  XVII 

tissue,  or  when  they  approach  one  another  and  come  together  in  order 
to  form  a  vascular  loop. 

The  phenomena  of  the  organism  which  bear  the  sharpest  impress 
of  their  physical  and  chemical  nature,  also  come  under  the  influence 
of  cellular  "sensations/7  Thus,  in  gastro-intestinal  digestion,  the 
secretion  of  the  active  juice  is  subordinated  to  the  control  of  the  nerve 
centres  and  even  of  the  psychic  centres.  The  sight  of  various  kinds 
of  food  stimulates,  unconsciously,  by  reflex  action  the  activity  of 
different  digestive  glands.  In  the  same  way  the  contraction  of  the 
contents  of  the  cells  of  a  plant  subjected  to  plasmolysis,  brings  about 
the  secretion  of  acid  in  order  to  augment  the  osmotic  pressure. 

Susceptibility,  whose  part  is  so  great  in  the  phenomena  of 
immunity,  taken  as  a  whole,  is  a  general  property  of  living  beings, 
regulated  by  a  common  law.  Thus,  in  the  chemiotaxis  of  the  lowest 
unicellular  organisms,  as  in  the  movements  and  the  osmotic  reaction 
of  plants,  there  is  manifested  the  same  psycho-physical  law  of  Weber- 
Fechner  which  regulates  our  own  sensations. 

All  cells  are  able,  by  modifying  their  function  under  the  direction 
of  susceptibility,  to  adapt  themselves  to  changes  in  the  surrounding 
conditions.  All  living  elements  are  able,  therefore,  to  acquire  a 
certain  degree  of  immunity.  But,  amongst  all  the  cells  of  the  animal 
body,  the  elements  which  have  retained  most  independence — the 
phagocytes — most  easily  and  first  acquire  immunity  to  infective 
diseases.  These  are  the  cells  which  betake  themselves  to  situations 
where  micro-organisms  and  their  poisons  make  their  appearance,  and 
which  manifest  a  reaction  against  them.  The  phagocytes  of  the 
immune  organism  ingest  and  destroy  micro-organisms  and  absorb 
toxins  and  other  poisons.  The  final  act  of  the  reaction  of  the 
phagocytes  is  constituted  by  the  chemical  or  chemico-physical  pro- 
cesses concerned  in  the  digestion  of  the  micro-organisms,  with  the 
help  of  cytases,  assisted  by  the  fixatives ;  in  the  defence  offered 
against  poisons  the  phagocytes  must  also  exert  a  chemical  action. 
[591]  Before  these  phenomena  come  into  play,  however,  the  phagocytes 
manifest  phenomena  which  are  purely  biological,  such  as  the  per- 
ception of  chemiotactic  and  other  sensations,  the  migration  towards 
menaced  situations,  the  ingestion  of  micro-organisms  and  the  absorp- 
tion of  toxins,  and  finally  the  secretion  of  substances  to  be  utilised  in 
intracellular  digestion. 

The  immunity  in  infective  diseases  presents  itself,  therefore,  as  a 
section  of  cellular  physiology,  and  especially  as  a  phenomenon  con- 


Summary  567 

cerned  in  the  absorption  of  micro-organisms.  This  absorption  being 
carried  out  by  an  act  of  intracellular  digestion,  the  study  of  immunity 
comes  into  the  chapter  on  digestion  regarded  from  the  general  point 
of  view. 

As  in  the  struggle  of  the  body  of  the  animal  against  infective 
agents  the  phagocytes  play  the  principal  part,  it  happens  that  in 
certain  diseases  the  micro-organisms  in  order  to  manifest  their 
morbific  effect  must  be  protected  from  the  attacks  of  these 
defensive  cells.  It  is  for  this  reason  that  the  cholera  vibrio, 
which  is  not  very  injurious  when  introduced  below  the  skin  of  the 
human  subject,  becomes  very  formidable  when  it  succeeds  in  gaining 
access  to  the  digestive  canal.  Incapable  of  maintaining  a  struggle 
against  the  phagocytes,  the  vibrio  is  able  to  overcome  in  the  stomach 
and  in  the  intestines  without  difficulty  the  obstacles  which.it  here 
meets  with.  It  is  for  this  reason  that  the  channel  of  entrance  of  the 
micro-organisms  at  times  plays  such  a  prominent  role  in  immunity 
against  infective  diseases. 

The  question  is  often  asked  whether  a  theoretical  study  of 
immunity  is  capable  of  rendering  service  in  the  search  for  means 
of  conferring  immunity  on  the  animal.  It  must  not  be  forgotten  that 
theory  and  practice  frequently  march  side  by  side,  but  that  sometimes 
they  advance  without  very  much  regard  for  each  other.  Thus  the 
first  preventive  inoculations  against  snake-bite,  small-pox,  and  pleuro- 
pneumonia,  attempted  by  laymen  were  evidently  made  independently 
of  any  theoretical  ideas  of  any  kind,  but  were  guided  by  the  purest 
empiricism.  On  the  other  hand,  the  theoretical  researches  on  the 
nature  and  origin  of  ferments  led  to  the  discovery  of  vaccinations 
by  means  of  micro-organisms  and  microbic  products  which  have 
rendered  immense  services  to  practical  medicine. 

The  discovery  of  antitoxins,  so  rich  in  practical  applications,  was 
influenced  by  theoretical  researches  on  the  mechanism  of  immunity. 
Yon  Behring  began  his  important  series  of  investigations  on  this 
subject  with  the  study  of  the  immunity  of  rats  against  the  anthrax 
bacillus.  It  did  not  suggest  itself  to  anyone  to  suppose  that  this  [592] 
question  could  have  the  slightest  immediate  practical  interest ;  never- 
theless, starting  from  this  investigation,  von  Behring,  after  giving  up 
the  theory  of  the  bactericidal  property  of  the  body  fluids  as  a  cause  of 
immunity,  advanced,  step  by  step,  to  the  discovery  of  the  antitoxic 
power  of  the  serums.  When  a  study  of  the  properties  of  the  blood  of 


568  Chapter  XVII 

animals  treated  with  the  red  corpuscles  of  another  species  was  com- 
menced, no  one  would  have  suspected  that  these  researches  would  end 
in  the  discovery  of  new  methods  for  the  recognition  of  human  blood  in 
medico-legal  researches,  or  in  the  interests  of  hygiene  for  the  determi- 
nation of  the  source  of  a  milk.  The  cellular  theory  of  immunity  is,  as 
yet,  of  too  recent  date  for  us  to  claim  the  right  to  expect  it  to  have 
amongst  its  assets  methods  for  purely  practical  application.  Never- 
theless, it  has  already  been  found  to  be  of  service  in  the  investigation 
of  problems  very  closely  affecting  medical  practice.  Lord  Lister,  the 
greatest  surgeon  of  the  nineteenth  century1,  asked  himself  how  it  was 
that  wounds  could  heal  "by  first  intention  under  circumstances  before 
incomprehensible.  Complete  primary  union  was  sometimes  seen  to 
take  place  in  wounds  treated  with  water-dressing,  that  is  to  say,  a 
piece  of  wet  lint  covered  with  a  layer  of  oiled  silk  to  keep  it  moist. 
This,  though  cleanly  when  applied,  was  invariably  putrid  within 
twenty-four  hours.  The  layer  of  blood  between  the  cut  surfaces  was 
thus  exposed  at  the  outlet  of  the  wound  to  a  most  potent  septic  focus. 
How  was  it  prevented  from  putrefying  as  it  would  have  done  under 
such  influence  if,  instead  of  being  between  divided  living  tissues,  it 
had  been  between  plates  of  glass  or  other  indifferent  material?" 
"  How  were  the  bacteria  of  putrefaction  kept  from  propagating  in  the 
decomposable  film  ?  Metchnikoff's  phagocytosis  supplied  the  answer. 
The  blood  between  the  lips  of  the  wound  became  rapidly  peopled 
with  phagocytes  which  kept  guard  against  the  putrefactive  microbes 
and  seized  them  as  they  endeavoured  to  enter.  If  phagocytosis  was 
ever  able  to  cope  with  septic  microbes  in  so  concentrated  and  intense 
a  form,  it  could  hardly  fail  to  deal  effectually  with  them  in  the  very 
[593]  mitigated  condition  in  which  they  are  present  in  the  air.  We  are  thus 
strongly  confirmed  in  our  conclusion  that  the  atmospheric  dust  may 
safely  be  disregarded  in  our  operations ;  and  Metchnikoff's  researches, 
while  they  have  illumined  the  whole  pathology  of  infective  diseases, 
have  beautifully  completed  the  theory  of  antiseptic  treatment  in 
surgery."  (Rep.  Brit.  Ass.,  p.  27.) 

We  may  even  attempt  to  increase  phagocytosis  in  surgical  opera- 
tions, especially  in  those  on  the  peritoneal  cavity,  by  there  setting  up 
an  artificial  aseptic  inflammation,  by  means  of  various  substances, 

1  "The  Relations  of  Clinical  Medicine  to  Modern  Scientific  Development,"  a 
discourse  delivered  at  Liverpool  in  September,  1896.  Rev.  scient.,  Paris,  1896, 
4e  se>.  t.  vi,  p.  481 ;  [Rep.  Brit.  Ass.  Adv.  Sci.,  London,  1896,  p.  3 ;  Brit.  Med. 
Journ.,  London,  1896,  Vol.  n,  p.  733]. 


Summary  569 

innocuous  in  themselves,  which  attract  a  large  number  of  leucocytes. 
In  laboratory  practice  this  method  is  in  daily  use  for  the  purpose  of 
increasing  the  resistance  of  an  animal  against  intraperitoneal  injections 
of  various  micro-organisms,  and  Durham  has  suggested  the  extension 
of  the  same  method  to  human  medicine.  Certain  surgeons  have 
already  made  attempts  in  this  direction. 

The  application  of  the  cellular  theory  of  immunity  to  researches 
on  new  micro-organisms  of  infective  diseases  has  already  been  crowned 
with  success.  Xocard  and  Roux  have  attempted  to  cultivate  in  the 
animal  body  the  virus  of  the  pleuropneumonia  of  cattle.  They  selected 
the  rabbit,  an  animal  naturally  refractory  against  this  infection.  On 
the  supposition  that,  in  this  immunity,  the  phagocytes  must  play  an 
important  part  as  destroyers  of  the  presumed  micro-organisms,  the 
idea  suggested  itself  to  them  to  withhold  the  virus  from  their  voracity. 
With  this  object  they  filled  sacs  of  collodion  or  of  reed  pith  with 
pleuropneumonia  virus,  and  introduced  these  sacs  into  the  peritoneal 
cavity  of  rabbits.  Some  time  after  this  operation  these  investigators 
were  able  to  demonstrate  in  the  contents  of  the  sacs  impregnated  by 
the  blood  fluid  of  rabbits,  immune  animals,  the  development  of 
specific  micro-organisms,  the  smallest  discovered  up  to  the  present. 
By  means  of  cultivations  of  this  micro-organism,  obtained  in  suitable 
media,  they  worked  out  a  method  of  vaccinating  animals  which,  as 
mentioned  in  Chapter  xv.,  has  already  begun  to  give  good  results  in 
veterinary  practice.  This  method  has  thus  contributed  to  the  pre- 
vention of  diseases,  a  branch  of  knowledge  which  has  made  such  great 
advances  since  medicine  became  an  exact  science  under  the  inspiration 
of  the  discoveries  and  ideas  of  Pasteur. 

Within  a  very  short  period  immunity  has  been  placed  in  possession 
not  only  of  a  host  of  medical  ideas  of  the  highest  importance,  but  also 
of  effective  means  of  combating  a  whole  series  of  maladies  of  the  most 
formidable  nature  in  man  and  the  domestic  animals.  Science  is  far  [594] 
from  having  said  its  last  word,  but  the  advances  already  made  are 
amply  sufficient  to  dispel  pessimism  in  so  far  as  this  has  been  sug- 
gested by  the  fear  of  diseases,  and  the  feeling  that  we  are  powerless 
to  struggle  against  them. 


LIST  OF  AUTHOEITIES  QUOTED. 


Abel,  443,  444,  445,  536 

Abel.    See  Loeffler 

Achalme,  96 

Achard  and  Bensaude,  264,  451 

Adil  Bey.    See  Nicolle 

Almquist,  178 

Arloing,  264,  452 

Arloing,  Cornevin  and  Thomas,  471 

Arnold,  411 

Arthus,  95 

Babes,  75,  348 
Bach,  408,  410 
Bail,  151,  185,  359 
Balbiani,  13,  23,  133 
Bardach,  150 
Barthels,  507 
Bary  (de),  31,  32 
Batzaroff,  411 

Baumgarten,  138,  193,  521,  522,  524 
Bayeux.     See  Roger 

Behring,  20,  152,  153,  205.  242,  290,  334, 
335,  348,  350,  352,  367,  369,  374,  375, 
378,  417,  526,  540.  561,  564,  567 

Behring  and  Kitasato,  266,  344,  347,  354, 
357,  493,  495 

Behring  and  Kitashima,  42, 290, 368, 370, 373 

Behring  and  Knorr,  355 

Behring  and  Nissen,  211,  226,  526,  531 

Bensaude,  439 

Bensaude.     See  Achard 

Bernard,  59 

Bernheim,  408 

Bertrand.     See  Phisalix 

Besredka,  111,  191,  231,  263,  273,  318, 353, 
390,  396 

Besson,  170 

Beumer  and  Peiper,  230 

Biedl  and  Kraus,  44 

Birch-Hirschfeld,  514 

Bitter,  525 

Bizzozero,  48,  177,  418,  423 

Bjelooussoff,  55 


Blagovestchensky,  323 

Bolton,  205 

Bordet,  22,  68,  79,  87,  90,  94,  95,  105, 107, 
111,  112,  115,  123,  166,  179,  185,  193, 
194,  196,  199,  215,  217,  223, 244, 251,  256, 
257,  258,  282,  298,  302,  313,  320,  321, 
535,  537 

Bordet.     See  Gengou 

Bordet  and  Danysz,  467 

Borrel,  478 

Borrel.     See  Roux,  Yersin 

Bouchard,  184,  232,  286,  323,  343,  427,  529 

Bouchard  and  Charrin,  42,  528 

Bourne,  327 

Braun,  12 

Brieger,  369 

Brieger  and  Frankel,  344 

Briot,  109 

Brticke,  66 

Brunner,  45 

Buchner,  87,  95,  184,  185,  188,  193,  255, 
357,  362,  377,  412,  512,  527,  528,  530, 
539,  540 

Cahanescu,  430 

Calmette,  334,  339,  345,  346,  347,  348,  358, 

365,  386,  389,  395,  425,  489 
Calmette.    See  Yersin 
Calmette  and  Dele"arde,  365 
Calmette  and  Salimbeni,  491 
Camus  and  Gley,  110,  121,  360 
Cantacuzene,  224,  225,  306 
Castle.    See  Davenport 
Cattani,  446 
Cayley,  484 
Celakovsky,  30 
Celli,  278 
Centanni,  446 
Chamberland,  470 
Chamberland.     See  Pasteur,  Roux 
Chantemesse,  259 

Chantemesse  and  Widal,  230,  207,  319,  437 
Chapeaux,  55,  56 


572 


List  of  Authorities  quoted 


Chart-in,  232,  286,  287,  427,  428,  541 

Charrin.     See  Bouchard 

Charrin  and  Gamaleia,  290,  343 

Charrin  and  Gley,  446 

Charrin  and  Lefevre,  419 

Charrin  and  Magnin,  427 

Charrin  and  Eoger,  232,  256 

Chatenay,  393 

Chauveau,  289,  446,  455,  511,  512 

Chepowalnikoff,  59 

Cherry.    See  Martin 

Cienkowski,  446 

Cobbett,  205 

Cohn,  23 

Colombot.    See  Sabraz&s 

Cornevin,  452 

Cornevin.     See  Arloing 

Couch,  53 

Courmont,  400 

Courmont.     See  Nicolas 

Courmont  and  Doyon,  330,  386,  394 

Curtis,  172 

Czaplewski,  146,  147 

Dallinger,  26 

Danysz,  21,  25 

Danysz.    See  Bordet 

Daremberg,  87 

Darwin,  8 

Davenport  and  Castle,  27 

Davenport  and  Neal,  24 

Decroly  and  Eousse,  396 

Deldarde.     See  Calmette 

Delezenne,  61,  96,  98,  107,  116 

Delezenne  and  Froin,  66 

Delius  and  Kolle,  277 

Dembinski,  147 

Denys,  533 

Denys  and  Havet,  151,  185 

Denys  and  Kaisin,  151 

Denys  and  Leclef,  243,  246,  283,  312 

Denys  and  Marchand,  313) 

Denys  and  van  de  Velde,  359 

Deutsch,  107,  293,  294,  537 

Dienert,  26 

Dieudonne,  139,  143,  147 

Dinkelspiel.    See  Nuttall 

Doederlein,  429 

Donitz,  391 

Dominici,  78 

Dominici.    See  Gilbert 

Doyon.    See  Courmont 

Dreyer,  350 

Duclaux,  26 

Dujardin-Beaumetz,  478 

Dungern  (von),  91,  109,  123,  324 

Durham,  256,  261,  569 

Dzierzgowsky,  448,  449 

Efifront,  26 

Ehrlich,  114,  115,  344,  346,  349,  356,  360 
361,  365,  378,  391,  392, 420,  449,  562,  563 
Ehrlich  and  Hiibener,  446,  452 


Ehrlich  and  Lazarus,  76 

Ehrlich   and   Morgenroth,   88,  89,  92,  95, 

104,  114, 116, 124,  193, 194,  199,  268,  537, 

538,  563 

Ehrlich,  Kossel  and  Wassermann,  496 
Ehrlich  and  Wassermann,  356 
Elmassian.     See  Morax 
Emden  (van),  264 
Emmerich,  237,  322,  527 
Emmerich  and  di  Mattei,  236,  527 
Emmerich  and  Low,  254 
Emmerich  and  Mastbaum,  475 
Ermengem,  420,  491 
Errera,  39 
Escherich.     See  Klemensiewicz 

Faber  (Knud),  344 

Fahrenholtz,  138 

Fehleisen,  434 

Fermi  and  Pernossi,  109 

Ferran,  480 

Fischer,  193,  213,  253 

Fischl  and  Wunschheim,  445 

Fleck,  413 

Fliigge,  43,  184,  525,  540 

Fodor,  184,  525 

Foerster,  380 

Fontana,  333 

Forssmann,  565 

Frank,  35,  154,  542 

Frankel,  344,  347,  499,  534 

Frankel.     See  Brieger 

Frankel  and  Sobernheim,  268 

Frantzius,  425 

Fraser,  345,  425 

Fredericq,  55,  57 

Freudenreich,  323 

Freund,  Grosz  and  Jelinek,  365 

Froin.    See  Delezenne 

Funck,  267,  319,  320,  456 

Galeotti.    See  Lustig 

Gamaleia,  419 

Gamaleia.     See  Charrin 

Gamier,  220,  304 

Gaule,  515 

Gautier,  400 

Gengou,  19,  20,  146,   151,  157,  185,  190, 

203,  242,  252,  255,  260,  264,  308,  543 
Gengou  and  Bordet,  190 
Geret.     See  Hahn 
Gheorghiewsky,  210,    234,    236,  261,  269, 

301,  307,  359 
Gibier,  137 
Giessler,  37 

Gilbert  and  Dominici,  424 
Gilkinet,  172 

Gley.    See  Camus,  Charrin 
Glogner,  434 
Goldschmidt,  411 
Gottstein,  499 
Gramatschikoff,  412 
Grancher.    See  Pasteur 


List  of  Authorities  quoted 


578 


Grawitz,  513,  515 
Griffon.     See  Landouzy 
Grosz.     See  Freund 
Gruber,  224,  256,  262,  542 
Gscheidlen.     See  Traube 
Guarnieri,  455 
Guinon.     See  Voisin 
Giinther,  541 

Haeckel,  517 

Haffkine,  480,  486-488 

Hafkine,  17 

Hahn,  188,  190 

Hahn  and  Geret,  197 

Hankin,  156,  187 

Hardy.     See  Kanthack 

Harnack,  337 

Haser,  507 

Havet.    See  Denys 

Hayem,  47,  514 

Hegeler,  196 

Herbst,  565 

Hericourt.    See  Richet 

Herzen,  62 

Hess,  144,  149,  524 

Hewlett.     See  Thomson 

Heymans.     See  Lang 

Heymans  and  Masoin,  396 

Hildebrandt,  109,  119,  412 

Himmel,  182 

Hippocrates,  342 

Hirsch  and  Mehring,  64 

Hoffmann  and  Recklinghausen,  46 

Horvath,  337 

Hiibener.     See  Ehrlich 

Hudalo,  436 

Hueppe,  254 

Hugenschmidt,  415 

Issaeff,  219,  262,  287,  318,  320,  441 
Issaeff.     See  Pfeiffer 

Jakowski,  42 
Jeanselme,  411 
Jelinek.     See  Freund 
Jenner,  507 
Jetter,  193 
Jona,  172 
Joubert.    See  Pasteur 

Kaisin.     See  Denya 

Kanthack,  360,  542 

Kanthack  and  Hardy,  185 

Karlinsky,  134,  260 

Kempner  and  Schepilewsky,  387 

Kempner.    See  Rabinowitsch 

Kilborne.    See  Smith 

Kitasato.     See  Behring 

Kitashima.     See  Behring 

Klebs,  514 

Klecki  (von),  44 

Klein,  324 

Klemensiewicz  and  Escherich,  443 


Klemperer,  271,  356,  411,  441,  449 

Klipstein,  170 

Knorr,  361,  362,  370,  375,  378,  383,  392r 

443 

Knorr.     See  Behring 
Koch,  137,  247,  278,  279,  283,  419,  425,  434, 

436,  466,  514,  529 
Kolle  and  Turner,  466,  467 
Kolle.    See  Delius,  Pfeiffer 
Kondratieff,  365 
Kossel,  110,  121,  183 
Kossel.    See  Ehrlich 
Kossiakoff,  25 

Kovalevsky,  41,  133,  134,  209 
Krafft-Ebing,  436 
Krajouchkine,  465 
Kraus  and  Seng,  258 
Kraus.     See  Biedl 
Kretz,  371 
Krikliwy,  46 
Krompecher,  83 
Kronig.    See  Menge 
Krukenberg,  30,  49,  55 
Kubler,  458 
Kupffer,  75 
Kuprianow,  204,  340 
Kurt,  499 

Laehr,  413 

Landonzy  and  Griffon,  451 

Landsteiner,  100 

Lang,  Heymans  and  Masoin,  363 

Langhans,  73,  84 

Laschtschenko,  188 

Laurent,  33,  35,  86 

Laveran  and  Mesnil,  173,  248,  316 

Lazarus,  272,  441 

Lazarus.    See  Ehrlich 

Leber,  79,  96 

Leclainche,  475,  476 

Leclainche.     See  Nocard 

Leclainche  and  Valtee,  107,  171,  472,  528 

Leclet     See  Denys 

Le  Dantec,  13 

Lefevre.     See  Charrin 

Leishman.     See  Wright 

Leo  and  Senator,  66 

Lepine,  564 

Lermoyez.    See  Wurtz 

Lesage,  47 

Le  Sourd.    See  Widal 

Leube,  67 

Levaditi,  223 

Levin,  159 

Lewes,  53 

Lewin,  337,  338 

Lignieres,  247,  279 

Lindemann,  68 

Lingelsheim,  193,  244,  312 

Lister,  521,  530,  568 

Loeffler,  7,  283,  513 

Loeffler  and  Abel,  267 

Lohr,  500 


574 


List  of  Authorities  quoted 


Lombard,  396 

London,  94 

Lorenz,  475 

Low.     See  Emmerich 

Lubarsch,  141,  151,  184,  529 

Lustig  and  Galeotti,  490 

Madsen,  349,  350 

Madsen.    See  Salomonsen 

Magnin.     See  Charrin 

Maksutow.     See  Pawlowsky 

Malm,  149 

Manfredi,  428 

Mankowski.     See  Podwyssozki 

Marchand,  167 

Marchand.     See  Denys 

Marchoux,  240,  276,  309,  311 

Marie,  331,  382,  465 

Marinesco,  75 

Marmorek,  243,  312 

Martel,  150,  159 

Martin  and  Cherry,  361 

Marx,  465,  476,  497 

Marx.     See  Pfeiffer 

Masoin.     See  Heymans,  Lang 

Massart,  34,  38,  39,  79,  281 

Mastbaum.     See  Emmerich 

Mattei  (di).     See  Emmerich 

Maupas,  16 

Mehring.     See  Hirsch 

Melkich.     See  Sawtchenko 

Mendez,  470 

Menge  and  Kronig,  429,  430 

Mesnil,  55,  75,  78,  135,  139,  141,  143,  188, 
209,  221,  238,  262,  270,  305,  307,  527 

Mesnil.     See  Laveran 

Metchnikofif,  31,  55,  69,  70,  73,  100,  101, 
116,  131, 137, 138, 146, 149, 151, 153, 154, 
156, 160, 163, 180, 181, 185,  214,  221,  227, 
237,  239,  241,  256,  259,  266,  271,  275,  286, 
287,  290,  302,  304,  311,  377,  382,  385,  393, 
396,  405,  426,  441,  520,  521,  522, 531,  532, 
534 

Metchnikoff  (Mme),  20,  159,  193 

Metin,  44 

Miller,  414,  415,  418 

Mitchell,  423 

Morax  and  Elmassian,  409 

Morgenroth,  109,  119,  331 

Morgenroth.     See  Ehrlich 

Morishima,  390 

Morse,  412 

Mouton,  15 

Moxter,  101,  185,  199 

MiiUer,  17,  89,  114,  233 

Myers,  68,  107 

Myers.     See  Stephens 

Neafr    See  Davenport 

N(§fedieff,  68 

Neisser,  194,  196 

Neisser  and  Wechsberg,  205,  298,  349,  359 

Nencki,  419,  424,  427 


Nencki  and  Sieber,  109,  355 

Nencki,  Sieber  and  Wyznikiewicz,  468 

Netter,  503 

Nicolas  and  Courmont,  353 

Nicolas,  Courmont  and  Prat,  353 

Nicolle  and  Adil  Bey,  279,  468 

Nikanoroff,  348 

Nissen.     See  Behring 

Nittis  (de),  277,  288 

Nocard,  148,  279,  494 

Nocard  and  Leclainche,  461 

Nocard  and  Eoux,  130,  466,  478,  479,  569 

Nolf,  94,  96 

Nowakowski,  12 

Nuttall,    107,    138,    150,    184,    192,    525, 

527 
Nuttall  and  Dinkelspiel,  107 

Oken,  337 
Opitz,  43,  44 
Oppel,  231 
Orlowski,  443,  444 

Pagel,  507 

Panum,  514 

Pasteur,  2, 181,  208,  288, 322,  477, 508,  510, 

511,  569 

Pasteur,  Chamberland  and  Eoux,  469 
Pasteur  and  Joubert,  144 
Pasteur,  Eoux  and  Grancher,  208 
Pasteur  and  Thuillier,  283,  473 
Patella,  97 

Pawloff,  59,  62,  65,  427 
Pawlowsky,  44,  323 
Pawlowsky  and  Maksutow,  348 
Peiper.    See  Beumer 
Pere,  26 

Pernossi.    See  Fermi 
Petruschky,  138 
Pfaundler,  259 
Pfeffer,  27,  38,  79 
Pfeiffer,  130,  165,  185,  219,  221,  267,  269, 

271, 277,  290,  301,  303,  320,  365,  438,  455, 

532,  533,  534 

Pfeiffer  and  Issaeff,  212,  533 
Pfeiffer  and  Kolle,  230,  267,  274,  302,  319, 

481 

Pfeiffer  and  Marx,  185,  264,  291,  442 
Pfeiffer  and  Proskauer,  253 
Phisalix,  387,  425 
Phisalix  and  Bertrand,  333,  337,  338,  345, 

347 

Pierallini,  218,  219 
Plato,  181 
Podwyssozki,  77 

Podwyssozki  and  Mankowski,  456 
Pollender,  11 
Ponfick,  46 
Portier,  96 
Prat.     See  Nicolas 
Preobrajensky,  431 
Prev6t,  374 
Proskauer.    See  Pfeiffer 


List  of  Authorities  quoted 


575 


Rabinowitsch  and  Kempner,  248,  316 

Eansom,  351,  379,  382,  389 

Ranvier,  409 

Rauchfuss,  501 

Recklinghausen  (von),  514 

Recklinghausen.     See  Hoffmann 

Remlinger,  447,  450 

Repin,  420 

Rhumbler,  15 

Ribbert,  413,  42S,  524 

Richet  and  Hericourt,  266,  532 

Rindfleiscb,  514 

Rochebrune  (de),  506 

Roden,  109 

Roger,  243,  257,  287 

Roger.     See  Charrin 

Roger  and  Bayeux,  414 

Rogers,  468 

Romer,  401 

Roncali,  170 

Roser,  515,  516 

Ross,  129 

Rossbach,  95 

Ronget.     See  Yaillard 

Rousse.     See  Decroly 

Roux,  156,  347,  358,  497,  498,  530 

Roux.     See  Nocard,  Pasteur 

Roux  (W.),  565 

Roux  and  Borrel,  340,  383,  386,  391 

Roux  and  Chamberland,  530 

Roux  and  Vaillard,  347,  355,  356,  357,  367, 

379,  432,  493 
Roux  and  Yersin,  343 
Ruffer,  427,  428,  523 
Rysselberghe  (van),  37,  39 

Sabouraud,  406 

Sabrazes  and  Colombot,  135 

Sakharoff,  160,  177 

Salimbeni,  222,  245,  261,  478 

Salimbeni.     See  Calmette 

Salmon,  455 

Salomon,  418 

Salomonsen,  19 

Salomonsen  and  Madseu,   346,  356,  370. 

379,  380 
Saltykoff,  272 
Samoiloff,  63 
Sanarelli,  262,  287,  415 
Sanchez-Toledo,  170 
Sarassewitch,  195 
Sawtchenko,  21,  99,   156,    162,    227,    240, 

260,  270 

Sawtchenko  and  Melkich,  162,  227 
Schaffer,  42 

Schattenfroh,  172,  188,  196 
Schepilewsky.     See  Kempner 
Schiff,  62 

Schimmelbusch,  42 
Schoumow-Simanowski.    See  Sieber 
Schumacher,  451 
Schutz,  283,  422 
Schutz.     See  Voges 


Schiitze,  107,  114 

Schiitze.    See  Wassermann 

Sclavo,  276,  310 

Selander,  290 

Senator.    See  Leo 

Seng.     See  Kraus 

Serpa  Pinto,  506 

Sicard.    See  Widal 

Sieber.    See  Nencki 

Sieber   and   Schoumow-Simanowski,    419, 

424 

Skchiwan,  172 
Slateano,  277 
Slawyk,  501 
Smith,  259 

Smith  and  Kilborne,  247,  279 
Sobernheim,  242,  276,  310,  441 
Sobernheim.     See  Frankel 
Soudakewitch,  75 
Soulie,  460 
Stadelmann,  97 
Stahl,  30,  31 
Stein,  12 

Stephens  and  Myers,  360 
Stern,  419,  542 
Sticker,  411 
Stohr,  428 

Stoudensky,  388,  394 
Strassman,  499 
Straus  and  Wurz,  417,  418 
Stroganoff,  429 

Takaki.    See  Wassermann 

Talma,  424 

Tarassewitch,  86,  87,  98,  99 

Tchistovitch,  68,  75, 106, 110, 120,  121,  122, 

283,  413 

Thiltges,  145,  147 
Thomas,  452 
Thomas.     See  Arloing 
Thomson  and  Hewlett,  410 
Thuillier.     See  Pasteur 
Tizzoni,  357,  446 
Tooth,  484 
Torday,  498 
Toussaint,  509 
Trapeznikoff,  139,  145 
Traube  and  Gscheidlen,  184 
Trommsdorff,  23,  189 
Trumpp,  261 
Turner.     See  Kolle 

Uhlenhuth,  68,  107 

Vaillard,  204,  335,  347,  356,  372,  447 

Vaillard.     See  Roux 

Vaillard  and  Rouget,  169,  170 

Vaillard  and  Vincent,  169,  394 

Vallee,  289,  425 

Vallee.     See  Leclainche 

Velde  (van  de).     See  Denys 

Viala,  465 

Vincent.    See  Vaillard 


576 


List  of  Authorities  quoted 


Yincenzi,  443 
Virchow,  48,  519,  524 
Voges,  238,  272 
Voges  and  Schiitz,  475 
Voisin  and  Guinon,  502 
Vries  (de),  35 

Wagner,  144 
Waldeyer,  514 
Wallgren,  168 
Walter,  64 
Walz,  193 
Warlomont,  456 
Washbourn,  485 

Wassermann,  115,  191,  205,  231,  234,  273, 
317,  318,  319,  322,  351,  358,  371,  441 
Wassermann.    See  Ehrlich 
Wassermann  and  Schiitze,  107 
Wassermann  and  Takaki,  292,  382,  394 
Wassilieff,  65 
Watson-Cheyne,  323 
Weber-Fechner,  27,  38,  566 
Wechsberg.     See  Neisser 
Wecker,  502 

Wehrmann,  417,  419,  424 
Weichhardt,  118,  124 


Weigert,  363,  379,  399,  424,  523 

Werigo,  281 

Wernicke,  276,  446,  447 

Widal,  257 

Widal.     See  Chantemesse 

Widal  and  Le  Sourd,  439 

Widal  and  Sicard,  260,  261,  264,  439,  440, 

450 

Wood.     See  Woodhead 
Woodhead  and  Wood,  323 
Wright,  482 

Wright  and  Leishman,  482 
Wunschheim.    See  Fischl 
Wurtz  and  Lermoyez,  410 
Wurz.     See  Straus 
Wyssokowitch,  43,  412,  485 
Wyznikiewicz.     See  Nencki 

Yersin,  468 

Yersin.     See  Eoux 

Yersin,  Borrel  and  Calmette,  487 

Zabolotny,  95 
Zeliony.     See  Zilberberg 
Ziegler,  519,  522 
Zilberberg  and  Zeliony,  282 


INDEX. 


Abrin,  344,  345,  346,  401 
Abrin  intoxication,  action  of  body  fluids  on, 
365,  420 ;    leucocvtic    reaction   against, 
393,  401 

Absorption.     See  Kesorption 
Acari,  mechanical  action  of,  3 
Acclimatisation.    See  Adaptation 
Acid  reaction  inside  phagocytes,  83,  182 
Acid,  secretion  of,  in  osmosis,  37,  566 
Acidophile  microbian  flora  of  stomach,  418 
Actinians,  digestion  in,  53,  82,  85 
Actinodiastase,  57,  197 
Actinophrys,  14,  18 
Adaptation.     See  also  Immunity 
Adaptation  to  toxic  substances,  21-27,  30, 
342,    390;    to    saline    solutions,    23,    30, 
515 ;    to  physical  conditions,  26,  30-31 ; 
of  plasmodia  to  arsenious  acid,  31 ;    of 
pancreatic  secretion  to  kind  of  food,  64, 
65 ;    of    phagocytes    to    destroy    micro- 
organisms, 281,  558,  566 ;  of  animals  to 
spinal  concussion,  etc.,  564;  of  cells,  513 
Addiment  (syn.  Complement),  95 
Agglutination  in  natural  immunity,  202, 
206;    and  phagocytosis,  202,  242,  245; 
in   the  diagnosis  of  typhoid,   256,  257, 
261,  439;    its  mechanism,   257;    of  red 
blood  corpuscles  by  serums,  258;  of  red 
blood  corpuscles  by  ricin,  360;  does  not 
prevent  growth  of  micro-organisms,  262 
Agglutinative      power,     transmission     by 
heredity  or  suckling,  450;  not  developed 
parallel  with  bactericidal  power,  483 
Agglutinins  in  immunity,  242,   245,  256- 
265,  295,  542,  559;    characters  of,  255, 
559;    origin  of,   in  immunised  animal, 
263-265,   294;    difference  between  fixa- 
tives and,  255,  265,  559;   not  the  same 
as  protective  substances,  268,  269,  294 
Albuminoid  substances,  resorption  of,  106- 

127 

Alexins.     See  also  Cytases 
Alexins,  87-95,  96,  98,  184,  193,  255,  528, 

533,  535,  539 

Alimentary  canal.  See  Intestine 
Alizarin  sulpho-acid,  13,  83,  183 
Alligator,  77,  143,  332,  401 


Amboceptors  (syn.  fixatives),  91,  93,  297, 
557 

Ammocoetes,  77,  78 

Amoeba,  14,  18,  23,  547,  549 

Amoebodiastase,  16,  197,  549 

Amoeboid  cells.  See  Leucocytes  and  Pha- 
gocytes 

Amphibia.     See  Frog,  Axolotl 

Amylase,  95;  in  the  urine,  65 

Androctonus.     See  Scorpion 

Anopheles  and  malaria,  129 

Antagonism  between  certain  bacteria,  323 

Anthrax,  11,  20,  21,  25,  41,  46, 180;  immu- 
nity of  dog  against,  149-151,  242;  acquir- 
ed immunity  of  Scolopendra  against,  209 ; 
natural  immunity  of  white  rat  against, 
526;  protective  serums  against,  20,  276, 
309-311;  phagolysis  in  acquired  immunity 
against,  280;  immunisation  against,  by 
means  of  other  bacteria,  323;  infection 
by  inhalation,  412;  by  ingestion,  423; 
immunity  against,  transmitted  to  off- 
spring, 445,  447;  vaccinations  against, 
208,  241,  468-471;  method,  470;  statis- 
tics, 471 ;  vaccination  against,  by  heated 
anthrax  blood,  507;  vaccines  against, 
208,  470,  509;  phagocytosis  in,  521, 
523 

Anthrax  bacillus,  action  on  rabies,  150; 
bactericidal  action  of  blood-serums  on, 
20,  146,  150,  151,  156,  157,  240;  in- 
creasing the  virulence  of,  150;  attenua- 
tion of,  208,  288;  eosinophile  transfor- 
mation in,  198;  protective  thickening  of 
bacterial  membrane  in,  242 ;  agglutination 
of,  203,  242,  260,  264 ;  natural  immunity 
against,  132-140,  143-147,  149-159,  511, 
512 ;  acquired  immunity  against,  239-242, 
276, 277 ;  antagonism  between,  and  certain 
bacteria,  323;  fate  of,  in  Algerian  sheep, 
512;  destruction  of ,  by  defibrinated  blood, 
525 

Anthrax,  symptomatic:  immunity  against 
bacilli  of,  171;  heredity  of  immunity 
against,  452;  vaccinations  against,  471- 
473;  phagocytosis  in,  523 

Antiabrin,  401 

37 


578 


Index 


Anti-arsenic  seruin,  390 

Anticytases,  112 

Anticytase  serum,  115,  371 

Anticytotoxins,  110,  118,  122,  127,  360 

Antidiastase,  109 

Antidiastatic  serums,  361 

Anti-enzymes,  109 

Antifixative,  112 

Antihaemolysins,  111 

Antihaemotoxins,  111,  119,  122 

Anti-infective.    See  Protective 

Antileucocidin,  359 

Antineurotoxin,  116 

Antirennet,  109 

Antiricin,  360 

Antisepsis,  Nature  replaces  by  asepsis,  432 

Antiseptics.  See  also  Toxins  and  Adaptation 

Antiseptics  and  foods,  26 

Antiseptic  action  of  the  gastric  juice,  417 

Antispermofixative,  124 

Antispermotoxins,  116,  122-126 

Antistreptococcic  serum,  243-245 

Antitetanin,  nervous  origin  of,  390 

Antitoxic.     See  also  Protective 

Antitoxic  unit  of  Ehrlich,  373,  496 ;  action 
of  non-specific  and  normal  serums  and  of 
broth,  365;  function  of  the  saliva,  417; 
function  of  pepsin  and  other  digestive 
ferments,  419,  424;  action  of  intestinal 
flora,  427 ;  property  of  the  body  fluids, 
531  (see  Body  fluids,  Serums) ;  power  of 
the  blood  of  new-born  children,  445 

Antitoxins,  natural,  in  normal  blood,  111, 
204,  444 ;  rarity  in  body  fluids  in  natural 
immunity,  204,  532,  533;  development 
of,  during  immunisation,  354 ;  properties 
of,  354;  present  in  various  fluids  of 
immunised  animal,  355,  531;  mode  of 
action  of,  on  toxins,  356-362.  371;  con- 
ditions acting  in  mixtures  of,  with  toxins, 
362;  immunity  against  toxins  not  in 
direct  constant  ratio  to  amount  of,  367- 
376;  effect  of  using  serum  from  same 
species,  379;  hypothesis  as  to  nature 
and  origin  of,  377-402,  562;  probable 
part  played  by  phagocytes  in  production 
of,  400-402;  rapid  regeneration  of,  after 
bleeding,  379;  augmentation  in  produc- 
tion of,  by  pilocarpin,  380;  transmission 
of,  by  milk  to  offspring,  449 ;  analogy 
of,  with  fixatives,  561 ;  hypersecretion  of, 
563 

Antivenomous  property  of  blood  of  scor- 
pion, 328;  action  of  serums,  334,  338; 
serum,  action  of,  334,  338,  358,  360 
Aqueous  humour,  bactericidal  action  of, 
184,192;  in  immunised  animals  contains 
no  fixative,  217,  222 ;  in  immunised 
animals  contains  antitoxin,  355 
Arsenic:  adaptation  to,  31,  343,  390;  pro- 
tective serum  against,  390;  leucocytic 
reaction  against,  396-399;  as  a  remedy 
against  microbial  disease,  513 


Arsenic  acid,  action  of,  on  anthrax  bacillus, 
25 

Arsenious  acid,  adaptation  of  plasmodia 
to,  31 

Arthropoda.  See  Clothes-moth,  Crayfish, 
Crustacea,  Daphnia,  Scolopendra,  Scor- 
pion, Spider,  Tick 

Arthrospores  of  Hueppe,  254 

Ascaris,  poor  microbian  flora  in  intestine 
of,  421;  phagocytic  organs  of,  547 

Asepsis  is  Nature's  method,  432 

Aspergillosis,  2,  4.     See  also  Mycoses 

Atrophic  diseases,  probably  due  to  a  para- 
site, 3 

Atropin,  reaction  of  rabbit  and  guinea-pig 
to,  395,  396 

Attenuation.  See  also  Vaccination,  Vac- 
cines 

Attenuation  of  micro-organisms  and  viruses, 
discovery  and  application  of,  208,  247, 
288,  508 ;  of  micro-organisms  by  the 
fluids  of  immunised  animals,  286-289; 
of  toxins,  344 

Autodigestion  in  yeast,  197 

Autospermotoxins,  101 

Autotoxins,  104 

Axolotl,  susceptible  to  tetanus  toxin, 
330 

Bacilli,  anaerobic,  natural  immunity 
against,  169,  170 

Bacillus  aerogenes,  agglutination  in,  264 

Bacillus  chauvaei.  See  Anthrax  sympto- 
matic 

Bacillus  coli  attacks  potato,  35;  vaccina- 
tion against,  267 ;  transformation  of,  into 
granules,  198 ;  modified  growth  on  certain 
serums,  259 

Bacillus  of  Doederlein,  429;  of  Kiel  water, 
408 

Bacillus  pyocyaneus,  42,  180,  254,  528; 
acquired  immunity  against,  210,  232- 
236,  301;  Pfeiffer's  phenomenon  in,  234, 
307;  special  forms  of  growth  in  serums 
from  vaccinated  animals,  256;  aggluti- 
nation of,  261,  307;  susceptibility  to  the 
toxins  of,  290,  351 ;  action  of  specific  se- 
rum on,  307,  358 ;  antagonistic  to  anthrax 
bacillus,  323 ;  immunisation  against  tos 
of,  351;  a  leucocidin  from,  359;  actk 
of  liver  on  toxin  of,  427;  heredity 
immunity  against  toxin  of,  446 

Bacillus  ranicida,  140 

Bacteria.     See  Micro-organisms 

Bactericidal  action  of  serum,  influence 
alkalinity  or  acidity  on,  196;    functi< 
of  the  tears,  408 

Bactericidal  property.  See  also  Body  fluids, 
Humoral  theory,  Serums 

Bactericidal  property:  in  blood  and  other 
fluids,  20,  146,  150,  151,  156,  157,  I' 
193,   211,   226,  233,  238,  240,  241, 
244,   512,   525-531,   542,   554;    of  boc 


579 


fluids,  theory  of  osmotic  pressure,  193, 
213 ;  of  extracts  of  glands  and  exuda- 
tions, 195;  of  the  saliva,  415;  absence 
of,  from  the  intestinal  ferments,  424,  567 ; 
of  serums,  Wright's  method  of  testing, 
483;  does  not  develop  parallel  with  ag- 
glutinative, 483  ;  and  immunity,  absence 
of  parallelism,  554 

Bactericidal  substance  (alexin,  comple- 
ment, cytase) :  in  blood  and  other  fluids, 
184-193,  534;  source  of,  in  body  fluids, 
185-193 ;  theory  of  leucocytic  secretions, 
187-191 ;  presence  in  body  fluids  due  to 
phagolysis,  191;  is  of  phagocytic  origin, 
185,  192;  in  body  fluids,  microphages 
source  of,  187;  not  resistant  to  heat, 
268;  and  so  distinguished  from  pro- 
tective substance,  268;  Pfeiffer's  theory 
of,  534 

Bacteriolysis.  See  Micro-organisms,  de- 
struction of 

Bacteriolysis,  analogy  between  haemolysis 
and,  537 

Bat,  immunity  against  tetanus  of  hiber- 
nating, 339 

Baumes-Colles'  law  in  syphilis,  436 

Behring's  "normal  serum,"  496 

Bile,  function  of,  60;  salts  protective 
against  snake  venom,  388;  protective 
function  of,  424 

Bipinnaria,  70,  518 

Blastomycetes.     See  also  Yeast-cells 

Blastomycetes,  resistance  of  Daphnia  to, 
131,  404,  520;  fate  of,  in  refractory 
organism,  172;  acidophile,  418 

Blood,  pepsin  in  the,  66,  563 ;  precipitins  in 
the,  68, 106, 107,  568;  fate  of  effusions  of, 
73;  bactericidal  power  of,  184  (see  also 
Bactericidal,  Serums) ;  natural  antitoxins 
in  normal,  111,  204,  444 ;  stimulant  (pro- 
tective) action  of  human,  271,  318; 
immunity  conferred  by  maternal,  447; 
recognition  of,  in  medico-legal  research, 
107,  568;  from  convalescents,  protective 
power  of,  437,  441,  443;  agglutination 
of  (see  Agglutination) 

Blood  corpuscles,  resorption  of  red,  47, 
50,  56,  57,  70,  72,  79-100,  537  (see  also 
Haemolysis) ;  fixation  of  cytase  by  red, 
194;  agglutination  of  red,  by  serums, 
258 ;  agglutination  of  red,  by  ricin,  360 
)dy  fluids.  See  also  Bactericidal,  Blood, 
Humoral  theory,  Serums 

y  fluids,  natural  immunity  and  the 
composition  of,  128-131,  146 ;  in  natural 
immunity,  absence  of  antitoxic  property 
in,  204;  bactericidal  power  of,  184-193, 
512,  525-531,  542  (see  also  Body  fluids, 
Serums) ;  antitoxic  power  of  the,  204,  531, 
533,  543;  protective  properties  of,  266-280 
oophilus  bovis,  247 

prdet's  sensibilising  substance,  91,  199, 
298,  535,  537,  557 


Botulism,  protective  action  of  fats  against 
toxin  of,  387;  action  of  digestive  dia- 
stases on  toxin  of,  420 

Bouchard's  theory  of  acquired  immunity, 
232, 286 ;  of  attenuating  power  of  serums, 
286-289 

Bouillon  de  panse,  473 

Bovidae,  acquired  immunity  of,  against 
Texas  fever,  247,  279;  protection  of, 
against  tetanus,  494;  vaccination  of, 
against  rinderpest,  425,  466-468;  against 
rabies,  466 ;  against  anthrax,  470 ;  against 
symptomatic  anthrax,  471 ;  against  pleu- 
ropneumonia,  477-479 ;  ancient  methods 
against  pleuropneumonia  in,  506 

Broth  as  a  protective  fluid,  320,  321,  365 

Buccal  cavity,  microbial  products  in  the 
protection  of  the,  416;  flora  of,  414 

Buchner's  theory  of  immunity,  512,  527 

Calf  lymph  vaccine,  method  of  prepara- 
tion, 456 

Carassius.     See  Goldfish 

Carmine,  fixation  of  tetanus  toxin  by, 
388,  394 

Cattle.     See  Bovidae 

Cattle  plague.    See  Einderpest 

Cayman.     See  Alligator 

Cellular  or  histogenic  immunity,  335,  336, 
340,  563-565 

Cellulosase,  86 

Cerebral  substance,  action  of  emulsions  of, 
on  toxins,  386 

Cerebral  tetanus,  383,  391 

Chemiotaxis.  See  also  Hyperleucocytosis, 
Susceptibility 

Chemiotaxis  in  Infusoria,  19 ;  in  plasmodia 
of  the  Myxomycetes,  30;  of  duodenal 
mucous  membrane,  64 ;  of  phagocytes, 
79,  108,  133,  167,  177,  280;  of  leucocytes 
for  rennet,  &c.,  119;  positive,  in  seg- 
mentation-cells of  frog  embryo,  565 

Cholera  antibody  (fixative),  253,  267,  292 

Cholera,  Asiatic,  protective  power  of  blood 
of  convalescents  from,  441;  vaccinations 
against,  480-481 

Cholera  peritonitis,  heredity  of  immunity 
against,  447,  448;  immunity  of  guinea- 
pig  against,  533 

Cholera  toxin,  alligator  resistant  to,  333 ; 
immunisation  against,  350;  action  of 
normal  serum  of  goat  on,  365 

Cholera  vibrio.  See  also  Pfeiffer's  pheno- 
menon, Vibrios 

Cholera  vibrio,  adaptation  of,  to  bactericidal 
substance,  23 ;  susceptibility  of  larva  of 
Ehiuoceros  beetle  to,  40,  133;  immunity 
of  frog  against,  142;  of  guinea-pig  against, 
163,  533;  extracellular  destruction  of, 
165,  212  (see  also  Pfeiffer's  phenomenon); 
eosinophile  transformation  in,  198;  ar- 
throspores  of,  254 ;  agglutination  of,  261, 
264;  protective  action  of  serums  against, 

37—2 


580 


Index 


268,  271,  318;  of  human  blood  against, 
271.  318 ;  immunity  to,  is  not  insuscepti- 
bility to  its  toxin,  290 ;  origin  of  protective 
property  against,  291 ;  protective  action 
of  various  fluids  against,  320;  antagonism 
between  certain  bacteria  and,  324;  in 
stomach,  419, 567 ;  susceptible  to  acids  in 
vitro,  419;  in  intestine,  423,  567;  serum 
from  animals  immunised  against,  532 

Cholesterin.     See  also  Fats 

Cholesterin,  fixation  of  toxins  by,  387 ;  fix- 
ation of  saponin  by,  389 

Chytridium,  12 

Cicatrisation  of  plants,  34 

Clasmatocytes,  78 

Clavelee  (la).     See  Sheep-pox 

Clavelisation  against  Sheep-pox,  460 

Clothes-moths,  micro-organisms  absent  from 
digestive  canal  of  larvae  of  certain,  420 

Coccobacillus  prodigiosus.  See  under  Micro- 
coccus 

Cockchafer  larva,  70,  326 

Complement  of  Ehrlich,  88,  91, 193,  251, 297 

Complementoids  of  Ehrlich,  115 

Concussion,  spinal,  adaptation  to,  564 

Conjunctiva,  elimination  of  micro-organisms 
by  the,  408 ;  absorption  of  toxins  by  the, 
409 

Copula  of  P.  Miiller,  91 

Cornea,  protective  resistance  by  the,  409 

Crayfish,  susceptible  to  certain  toxins,  345; 
blood  of,  antitoxic  against  scorpion 
venom,  366 ;  poor  intestinal  flora  of,  421 

Crickets  and  micro-organisms,  41,  133; 
natural  immunity  against  toxins  in,  329 

Crustacea.     See  Crayfish,  Daphnia 

Crustacea,  protective  function  of  integument 
of,  404 

Cyprinus.    See  Goldfish 

Cytase  of  Laurent,  86 

Cytases  (syn.  alexins,  complements),  93,  98, 
123;  elaborated  by  phagocytes,  197,  252, 
539,  549-556 ;  thrown  out  into  plasmas 
during  phagolysis,  95,  99,  102,  197,  252, 
551-554;  bactericidal  power  of ,  183,  184, 
191,  193-198,  217  (see  also  Bactericidal, 
Body  fluids,  Serums) ;  unity  or  plurality 
of,  in  same  serum,  193, 197;  absorption  of, 
194,  200 ;  two  kinds  of,  macrocy tase  and 
microcytase,  195,  296,  549;  characters  of 
the,  197,  549;  enzymes  other  than,  in 
phagocytes,  197;  in  the  immunised  or- 
ganism, 250-255,  296,  317,  554;  presence 
or  absence  of,  how  determined,  253 ;  Ehr- 
lich's  and  author's  views  on,  contrasted, 
297 ;  compared  with  fixatives,  555 

Cytotoxins,  105  (note),  110,  116 

Daphnia,  resistance  of,  to  Blastomycetes, 

131,  404,  520 

Darwin  on  the  extinction  of  the  elephant,  8 
Dennis,  arrest  of  micro-organisms  in  the, 

406 


Desmon  (of  London),  91 

Diastases.  See  Digestive  ferments,  Ferments 

Digestion  in  the  higher  animals,  49,  59-65; 
psychical  and  nervous  elements  in,  62, 
566;  extracellular,  by  secreted  juices,  49, 
58,  62 ;  the  liver  of  the  Mollusca  as  second 
organ  of,  59;  in  the  tissues,  67;  and  re- 
sorption  closely  related,  69,  85;  by  ma- 
crophagic  organs,  85,  150 

Digestion,  intracellular.  See  also  Phago- 
cytes, Phagocytosis,  Eesorption 

Digestion,  intracellular,  48,  85,  517,  518, 
520;  in  the  Protozoa,  13,  30,  49;  in 
Planarians,  49,  71,  82 ;  in  Actinians,  53, 
82,  85;  in  Sponges,  69,  517;  transition 
from,  to  digestion  by  secreted  juices,  49, 
58 

Digestive  ferments,  antitoxic  function  of, 
424  ;  action  of,  on  toxin  of  botulism,  420 

Diphtheria,  7,  41, 132,  204 ;  antitoxic  power 
of  blood  of  convalescents  from,  443 ;  anti- 
toxic power  against,  in  blood  of  healthy 
persons,  444;  and  in  blood  of  new-born 
children,  445;  heredity  of  immunity 
against,  445,  447,  448;  influence  of  anti- 
cytase  serum  on,  371 ;  vaccinations  against, 
495-503;  serum  against,  495;  standard- 
isation and  testing  of  this  serum,  496- 
498;  its  protective  and  antitoxic  powers 
do  not  develop  in  equal  ratio,  497;  its 
prophylactic  use,  498-503;  accidents 
during  treatment,  499,  502;  statistics, 
500-503 

Diphtheria  toxin,  increased  susceptibility  of 
immunised  guinea-pig  to,  290;  natural  im- 
munity of  rat  and  mouse  against,  204,  339 ; 
natural  immunity  of  frog  against,  330; 
immunisation  against,  344,  347,  349,  353 ; 
attenuation  of,  344 ;  preventive  action  of 
nucleohiston  on,  365;  action  of,  on  brain 
of  laboratory  animals,  386 ;  sets  up  local 
lesions  in  the  conjunctiva,  409;  pepsin 
destroys,  419 

Diplococcus  pneumoniae.  See  Pueumococ- 
cus 

Diseases,  fear  of,  and  pessimism^  1,  569 ; 
atrophic,  probably  due  to  a  parasite, 
3;  mechanical  element  as  etiological 
factor,  3;  toxic  element  as  etiological 
factor,  4 ;  developed  on  the  earth  at  a  very 
early  epoch,  8;  and  extinction  of  species, 
8;  infective,  in  multicellular  plants,  29- 
39 ;  set  up  by  Fungi.  See  Fungi 

Dog,  immunity  of,  against  anthrax,  149- 
151,  242;  action  of  anthrax  bacillus  on 
rabid,  150;  immunity  of,  against  strepto- 
cocci, 167;  naturally  refractory  against 
a  staphylococcus,  266 ;  bactericidal  action 
of  blood  of,  on  anthrax  bacillus,  150, 151, 
156;  digestion  of  gelatine  by  leucocytes 
of,  108 ;  enterokynase  in  lymphoid  organs] 
of,  61;  digestive  fluids  of,  62-65;  dism 
fecting  power  of  small  intestine  of,  422 


Index 


581 


phagocytosis  in,  149,  151;  haematozoon 
in,  279 

Domestic  animals,  immunisation  of,  against 
disease.  See  Bovidae,  Dog,  Goat,  Horse, 
Pig,  Sheep,  Swine,  Vaccines,  Vaccinations 

Dourine,  2,  247 

Drepanidium,  515 

Drugs,  absorption  of,  by  leucocytes,  400 

Duodenum,  chemiotaxis  of  mucous  mem- 
brane of,  64 

"Dust"  cells,  75,  411-414 

Eel's  serum.     See  also  Ichthyotoxin 

Eel's  serum,  toxic  action  of,  20,  111,  563; 
and  precipitins,  68,  106 

Effusions  of  blood,  fate  of,  73 

Ehrlich's  neutral  red  reaction,  13,  83,  181; 
classification  of  leucocytes,  74,  76-78; 
theory  of  side-chains  or  receptors,  120, 
381-384,  538,  557,  562-563;  compared 
with  theory  of  phagocytes,  296-299,  538, 
558;  "immunising  unit,"  373,  496 

Elephant,  extinction  of,  8 

Elimination  of  micro-organisms  from  the 
body,  43,  46 ;  by  the  epidermis,  406 ;  by 
the  conjunctiva,  408;  by  the  nasal  mu- 
cosa,  410 

Emys.     See  Turtle 

Endo-enzymes,  197 

Endotrypsin  of  yeast,  197 

Enterokynase,  59,  98 

Enzymes.     See  Ferments 

Eosinophile  leucocytes,  secretion  by,  in 
bacteriolysis,  187,  542 

Eosinophile  staining  reaction,  198 

Epidermis,  exfoliation  of  the,  406 

Ernst's  bacillus,  immunity  of  frog  against, 
140 

Erysipelas.    See  Swine  erysipelas 

Erysipelas,  immunity  in,  434 

Erysipelas  streptococcus,  protective  action 
of,  against  anthrax,  323;  its  use  in  ma- 
lignant tumours,  434 

Excretion.     See  also  Elimination 

Excretion  in  relation  to  micro-organisms, 
43,  432 ;  of  pepsin  in  the  urine,  65 ;  of 
pepsin  in  the  blood,  66,  563 

Exfoliation  of  the  epidermis,  406 

Exudations,  bactericidal  power  of,  185, 193, 
195 

Farcy,  slow  evolution  of,  406 

Fats,  protective  action  of,  against  toxins, 
387 

Ferments.  See  also  Intestinal,  Digestive, 
Fibrin-ferment,  Gastric  juice,  Saliva, 
Trypsin 

Ferments,  Pasteur  on  the  organised  nature 
of,  2;  soluble  (diastases  or  enzymes),  in 
digestion,  49,  55,  57,  108,  109,  197;  anti- 
toxic function  of  digestive,  424;  phago- 
cytic,  197,  549-559;  hypersecretion  of,  563 

Fibrin  ferment  (plasmase),  95,  197,  550 


Fishes.     See  Goldfish 

Fishes,  phagocytosis  in,  135 

Fixatives  (immunising  body,  or  amboceptor, 
or  sensibilising  substance),  88,  92-95, 
97,  98,  103-105,  199-202,  296;  synonyms 
of,  91 ;  analogy  of,  with  enterokynase, 
98;  presence  of,  in  plasmas,  103,  112- 
114,  217 ;  in  protective  serums,  269,  438  ; 
in  mesenteric  glands,  98 ;  in  spenno- 
toxins,  101;  origin  of,  103,  294,  537, 
556-559  ;  specificity  of,  88, 105,  216,  251, 
253,  296;  rarity  of,  in  normal  fluids, 
199-201,  250;  method  of  determining 
whether  present  in  a  serum,  199 ;  absent 
from  aqueous  humour  of  immunised 
animals,  217,  222,  251;  in  the  immu- 
nised organism,  250-255;  properties 
of,  251,  253,  255,  554;  differ  from 
agglutinative  substances,  255,  265,  559 ; 
relation  of,  to  phagocytosis,  291,  295 ; 
part  played  by,  in  Pfeiffer's  pheiiomenon, 
251,  295 ;  and  protective  substances 
closely  connected,  269,  294,  295,  561; 
compared  with  cytases,  555 ;  mechanism 
of  action  of,  557 

Food  substances,  absorption  of,  by  other 
channel  than  alimentary  canal,  67 

Foods  and  antiseptics,  26 

Foreign  bodies,  fate  of,  in  organism,  46, 
52,  55,  56,  517 

Formed  elements,  resorption  of  the,  47, 
67-105 

Fowl,  immunity  of,  against  anthrax,  144, 
159 ;  phagocytosis  in,  144,  282 ;  bacteri- 
cidal action  of  plasma  of,  on  anthrax, 
146 ;  blood  serum  of,  and  tetanus,  204 ; 
immunity  of,  against  tetanus,  204;  na- 
tural immunity  of,  against  tetanus  toxin, 
335 ;  influence  of  removal  of  parts  of 
brain  and  cord  on  tetanus  in,  384 

Fowl  cholera,  infection  of  laboratory  ani- 
mals with,  181 ;  vaccine  against,  208 ; 
phagocytosis  in,  282 ;  action  of  exuda- 
tions of  fowls  vaccinated  against,  288; 
acquired  immunity  against,  288,  508 ; 
failure  of  bacillus  of,  to  grow  in  certain 
media,  510 

Friedlander's  bacillus  prevents  infection  by 
anthrax,  323 

Frog,  phagocytosis  in,  137,  142  ;  immunity 
of,  against  anthrax,  137  ;  against  Ernst's 
bacillus,  140;  against  bacillus  of  mouse 
septicaemia,  141 ;  against  cholera  vibrio, 
142 ;  acquired  immunity  of,  against  pyo- 
cyanic  disease,  210,  301 ;  natural  im- 
munity of,  against  tetanus  toxin,  330 ; 
against  diphtheria  toxin,  330;  immuni- 
sation of,  against  abrin,  345  ;  absorption 
of  tetanus  toxin  by  brain  of,  386 

Frog  embryo,  positive  chemiotaxis  in  seg- 
mentation-cells of,  565 

Fungi,  diseases  set  up  by,  2,  4,  18,  32,  131, 
135,  404  (see  also  Aspergillosis,  Mycoses) 


582 


Index 


Galactose.     See  Milk-sugar 

Gamaleia's  vibrio.    See  Vibrio  metchnikovi 

Gastric  juice,  antiseptic  action  of,  417; 
psychic  influence  on,  63,  566.  See  Pepsin 

Gelatine,  resorption  of,  107 

Gentilly  bacillus.     See  Pneumo-enteritis 

Gerbil,  tubercle  in,  22,  183 

Goat,  action  of  normal  serum  of,  on  cholera 
toxin,  365 ;  vaccination  of,  against  rabies, 
466  ;  acquired  immunity  in,  563 

Goldfish,  72,  135 

Goose  septicaemia.  See  Spirochaete  an- 
serina 

"  Greek  method"  of  variolisation  against 
small-pox,  507 

Gruber's  theory  of  immunity,  256,  262 

Guinea-pig,  immunity  of,  against  spirilla, 
160,  162;  against  vibrios,  163,  211-227, 
275,  287,  531,  533 ;  against  streptococci, 
165 ;  against  tetanus  bacillus,  169 ;  against 
symptomatic  anthrax,  171 ;  against  Try- 
panosomata,  173 ;  acquired  immunity 
against  spirilla  of  recurrent  fever,  227- 
230;  against  typhoid,  191,230;  against 
Bacillus  pyocyaneus,  234-236 ;  against 
anthrax,  276,  277 ;  phagocytosis  in,  162, 
163,  166,  170,  223;  hypersusceptibility 
of  immunised,  to  diphtheria  toxin,  290 ; 
protective  power  of  serum  of  immunised, 
293  ;  effect  of  removal  of  spleen  of,  293  ; 
antivenomous  action  of  serum  of,  338 ; 
immunisation  of,  against  cholera  toxin, 
351 ;  increasing  natural  susceptibility  of, 
to  toxins,  369,  370;  reaction  of,  to 
atropin,  396 

Haematopoietic  organs.   See  also  Lymphoid 

organs 

Haematopoietic  organs  as  source  of  pro- 
tective substance,  292-294 
Haematozoa.  See  Piroplasma,  Trypanosoma 
Haematozoon  in  dog  closely  allied  to  that 

of  Texas  fever,  279 
Haemolysis.      See  also  Blood   corpuscles, 

resorption  of 
Haemolysis,  79-100,   111,   112,   537;    the 

two   substances  which    act    in,   88,   98, 

538 ;  analogy  between  bacteriolysis  and, 

537 

Haemomacrophages,  76,  136 
Haptophore  atomic  group  in  a  toxin,  120, 

350,  384 
Hedgehog,   natural  immunity  of,   against 

poisons  and  venoms,  337 
Helix  pomatia,  70,  134 
Heredity  of  immunity,  445-453,  513 
Herpestes.     See  Mongoose 
Hibernation,  effects  on  resistance  to  toxins, 

339 

Hippocampus,  135 
Histogenic  immunity,  336  (tee  Immunity, 

cellular) 
Hog  cholera,  resemblance  of  bacillus   of, 


to  that  of  pneumo-enteritis,  259  ;  serum 
of  animals  vaccinated  against,  260;  ag- 
glutination in,  260;  protective  action  of 
serums  against,  272;  susceptibility  of 
vaccinated  animals  to  the  toxin,  290 

Horse.     See  also  Diphtheria 

Horse,  acquired  immunity  against  cholera 
vibrio,  222 ;  against  streptococci,  244, 
245,  313 ;  local  reaction  to  tetanus  toxin 
in,  352 ;  immunised,  with  poor  yield  in 
antitoxin,  373,  375  ;  reaction  of,  to  one 
unit  of  toxin,  378 ;  antitoxic  power  of 
serum  of  normal,  380 ;  phagocytosis  in, 
245,  313 ;  antivenomous  action  of  serum 
of,  338  ;  vaccination  of,  against  rabies, 
466 ;  vaccination  of,  against  anthrax, 
470 ;  protective  serum  against  tetanus 
in,  493 

Humoral  phenomena  in  immunity,  184, 
250,  290,  437-440,  525-531,  542,  543 

Humoral  theories  of  immunity,  184,  525- 
531,  542,  543  ;  attempts  to  reconcile  with 
theory  of  phagocytes,  539 

Humours.     See  Body  fluids,  Serums 

Hyperleucocytosis.     See  also  Chemiotaxis 

Hyperleucocytosis  during  immunisation, 
352,  393 

Hypersecretion,  563  (see  Eeceptors) 

Hypersusceptibility  to  toxins  in  immunised 
animals,  290,  368-374,  564 

Hyphomycetes,  diseases  caused  by,  2 

Hypopyon,  pus  of,  96 

Ichthyotoxin,  110,  120,  121,  122,  326,  360 
(see  also  Eel's  serum) 

Immunisation.  See  Immunity,  acquired, 
artificial  and  temporary,  Vaccination 

Immunisation  against  toxins,  principal 
methods  of,  345-350 ;  by  unmodified 
toxins,  345-346 ;  by  modified  toxins, 
347 ;  by  mixtures  of  toxin  and  antitoxin, 
348 ;  by  toxones  and  toxoids,  349 ;  phe- 
nomena produced  during,  352-354 

Immunising  body  of  Ehrlich,  91,  251 ; 
unit  of  Ehrlich,  373,  496 

Immunity,  historical  sketch,  505-543 ; 
summary,  544-569 ;  by  attenuated  micro- 
organisms, 2 ;  predisposition  or  absence 
of,  7  ;  against  infective  diseases,  9 ;  de- 
finition of,  10 ;  against  micro-organisms, 
10,  41,  42,  128-206,  207-324;  against 
toxins,  10,  41,  42,  325-341,  342-402; 
not  same  as  against  micro-organisms, 
290,  351 ;  in  unicellular  organisms,  11- 
28;  in  multicellular  plants,  29-39;  in 
plants,  action  of  manures  on,  36  ;  in  the 
animal  kingdom,  40-66  ;  cellular  or  histo- 
genic,  335,  336,  340,  563-565;  active 
(Ehrlich),  378  =  isopathic  immunity  (von 
Behring);  passive  (Ehrlich),  378,  453 
=  antitoxic  immunity  (von  Behring) ; 
passive  against  micro-organisms,  300- 
324,  560 ;  isopathic  (von  Behring),  378 ; 


Index 


583 


antitoxic  (von  Behring),  378  ;  of  the  skin, 
403-407 ;  of  the  mucous  membranes, 
407-432;  susceptibility  in,  565  (see  also 
Hypersusceptibility,  Susceptibility);  chan- 
nel of  entrance  in,  567 ;  applications  of 
theory  of,  to  medical  practice  and  to  the 
research  of  new  organisms,  567-569 

Immunity,  natural :  10, 17, 18,  30 ;  amongst 
Invertebrates,  40,  131-135 ;  amongst 
Vertebrata,  41,  135-174;  against  micro- 
organisms, 128-174,  175-206;  and  com- 
position of  body  fluids,  128-131 ;  against 
anaerobic  bacteria,  169, 170 ;  part  played 
by  inflammation  in,  176 ;  importance  of 
microphages  in,  177 ;  humoral  theory  of, 
184 ;  agglutination  in,  202,  206 ;  against 
toxins,  325-341 

Immunity,  acquired  :  10,  19,  31 ;  against 
micro-organisms,  207-249,  250-299; 
against  vibrios,  211-227 ;  against  pyo- 
cyanic  disease,  210, 232-236, 301 ;  against 
spirilla  of  recurrent  fever,  227-230; 
against  typhoid  bacillus,  230 ;  against 
swine  erysipelas,  236-239 ;  against  an- 
thrax, 239-242 ;  against  streptococcus, 
243-247;  against  Trypanosomata,  247- 
249,  316  ;  against  staphylococcus,  266 

Immunity,  rapid  and  temporary:  against 
micro-organisms,  300-324 ;  conferred  by 
specific  serums,  301-317 ;  conferred  by 
normal  serums,  317-320 ;  conferred  by 
fluids  other  than  serums,  320-322 ;  con- 
ferred by  non-specific  micro-organisms, 
322-324 

Immunity,  artificial,  against  toxins,  342- 
402 ;  against  bacterial  toxins,  343 ; 
against  vegetable  toxins,  344, 365 ;  against 
snake  venom,  345;  not  in  direct  ratio 
to  amount  of  antitoxin  in  body  fluids, 
367-376 

Immunity  acquired  by  natural  means, 
433-453  ;  acquired  after  recovery  from 
infective  diseases,  433-444 ;  acquired  by 
heredity,  445-453;  conferred  by  maternal 
blood,  447;  by  the  yolk,  449;  by  the  milk 
of  the  mother,  449 

Immunity,  acquired:  amongst  In vertebrata, 
209-210  ;  amongst  Vertebrata,  210-249  ; 
relation  of  Pfeiffer's  phenomenon  to, 
224;  Bouchard's  theory  of,  232,  286; 
double  action  of  cytases  and  fixatives 
in,  250-255,  296,  554 ;  agglutinative  sub- 
stances in, 242, 245, 256-265, 295, 542, 559 ; 
protective  properties  of  body  fluids  in,  266- 
280;  phagocytosis  in,  220,  223-226,  245, 
280-286,  295;  origin  of  fixative  properties 
in  body  fluids  in,  294 ;  relation  between 
fixatives  and  phagocytosis  in,  291,  295 ; 
humoral  phenomena  in,  184,  250,  290, 
525-531,  542,  543 ;  bactericidal  power  of 
fluids  in,  250;  Gruber's  theory  of,  256, 
262 ;  against  micro-organisms,  suscepti- 
bility to  the  specific  toxin  in,  289 ; 


principal  phenomena  associated  with, 
295-296 ;  against  micro-organisms  in  no 
ratio  to  protective  power  of  blood,  372- 
374 ;  by  suckling,  mouse  the  only  animal 
in  which,  450,  452  ;  theory  of  exhaustion 
of  nutrient  medium  as  cause  of,  510- 
512;  theory  of  presence  of  inhibitory 
substance,  511,  512 ;  theory  of  local  in- 
flammatory reaction,  512 ;  theory  of 
adaptation  of  cells  in,  513 ;  theory  of 
phagocytes  in,  514-525,  539-543  ;  theory 
of  bactericidal  power  of  body  fluids,  525- 
531,  542,  543 ;  theory  of  antitoxic  power 
of  body  fluids,  531 ;  theory  of  extra- 
cellular destruction  of  micro-organisms 
by  leucocytic  secretions,  187-191,  533- 
537,  542;  theory  of  side-chains,  120, 
381-384,  538,  557,  562-563;  present 
phase  of  the  question  of,  540-543 

Immunproteidin  of  Emmerich  and  Low, 
254 

Infection,  agents,  mechanical  and  other, 
that  prevent  or  aid,  3-5,  170-173,  426 
(see  also  Diseases,  Elimination,  Micro- 
organisms) 

Inflammation  in  immunity,  176,  512 ; 
Cohnheim  on,  518;  and  phagocytosis, 
516,  519-520,  547,  568 

Influenza  bacillus,  cultivation  of,  in  body 
fluids,  130,  554 ;  vaccination  against,  277 

Infusoria.     See  also  Trypanosoma 

Infusoria,  12-20,  23,  26,  326 

Inoculation.  See  Immunisation,  Vaccina- 
tion 

Insects,  natural  immunity  in,  132,  326, 
329 ;  acquired  immunity  in,  209 ;  pro- 
tective lining  of  digestive  canal  of,  421 

Insusceptibility  of  cells  of  refractory  ani- 
mals, 341 

Integument  of  Invertebrata,  protective 
function  of,  404 

Intermediary  body,  88,  91,  296,  557 

Intestine,  protective  function  of  the,  422; 
microbian  flora  of,  420 ;  antitoxic  action 
of  this  flora,  427 

Intestinal  ferments,  absence  of  microbi- 
cidal  power  from,  424,  567 ;  intestinal 
micro-organisms,  favouring  and  retarding 
functions  of,  426 ;  destruction  of  toxins 
by,  427 

Invertebrata,  natural  immunity  in  the,  40, 
131-135,  326-329;  acquired  immunity 
in  the,  209-210,  301 ;  immunisation  of, 
by  specific  serums,  301 ;  protective  func- 
tion of  integument  of,  404 

Iodine  trichloride  in  immunisation,  347 

Iron,  absorption  of,  by  leucocytes,  399 

Irritability,  part  played  by,  18,  27  (see 
also  Susceptibility) ;  in  plants,  38 

Isaria,  resistance  to  infection  by,  329 

Koch's  phenomenon  in  tuberculosis,  437 
Kupffer's  cells,  75 


584 


Index 


Leprosy,  etiological  factors  in,  4 

Leprosy  bacillus,  75,  411 

Leucocidin,  and  its  neutralisation,  359 

Leucocytes.     See  also  Phagocytes 

Leucocytes  (amoeboid  cells)  in  resorption, 
47,  73,  175,  514,  515 ;  adaptation  of,  to 
virulent  bacteria,  an  education,  281; 
various  categories  of,  74-79  ;  soluble  fer- 
ments of,  95 ;  chemiotaxis  of,  119,  177  ; 
theory  of  bactericidal  secretions  by,  187- 
191,  533-537,  539,  540,  542;  action  of 
leucocidin  on,  359  ;  absorption  of  poisons 
by,  393-400;  situations  where  there  are 
no  pre-existing,  551 

Lily  of  the  valley,  acquired  immunity  in, 
513,  515 

Liver,  serum  against  cytotoxin  acting  on, 
116 ;  protective  function  of  the,  427  ; 
of  Mollusca  an  organ  of  second  diges- 
tion, 59 

Lizard,  resistance  of,  to  tetanus  toxin, 
332 

Lugol's  solution  in  immunisation,  347 

Lupus,  slow  growth  of,  406 

Lymphocytes.  See  also  Leucocytes,  Phago- 
cytes 

Lymphocytes,  76,  78 

Lymphoid  organs.  See  also  Haemato- 
poietic organs,  Phagocytic  organs 

Lymphoid  organs,  protective  function  of 
the,  428 ;  as  source  of  sensibilising  sub- 
stance (fixative),  537 

Lymphomacrophages,  76 

Macrocytase  (alexin,  complement),  86,  98, 
105,  112,  196,  549;  analogy  of,  with 
actinodiastase,  86  ;  escape  of,  during  pha- 
golysis,  95,  99,  102,  552  ;  presence  of,  in 
spermotoxin,  101 ;  origin  of,  103  ;  active 
for  resorption  of  animal  cells,  196,  197, 
296;  in  extracellular  solution  of  red 
corpuscles,  552 

Macrophages,  76,  77,  79,  547;  the  part 
they  play  in  resorption,  80-100,  176; 
staining  reactions  of,  77;  in  phagocytosis, 
144,  148,  154,  157,  161,  162,  164,  173, 
184,  228,  245,  321,  548 ;  act  more  especi- 
ally in  resorption  of  animal  cells,  176, 
196,  548  ;  but  intervene  specially  against 
human  tubercle  bacillus  in  pigeon,  148 ; 
against  spirilla,  162, 177,  228 ;  and  against 
streptococci,  245  ;  not  source  of  bacteri- 
cidal substance  in  body  fluids,  187 ;  part 
played  by,  in  arsenic  poisoning,  397  ;  the 
principal  source  of  antitoxin,  401;  of 
skin,  reaction  of,  against  micro-organ- 
isms, 407 

Macrophagic  organs,  digestive  property  of, 

Malaria,  immunity  against,  129,  278 ;  pro- 
tective action  of  serum  in,  278;  im- 
munity acquired  after,  434 

Manures,  influence  on  plant  diseases,  36 


Marmot,  immunity  of  hibernating,  against 
tetanus,  339 

Martin's  broth  (bouillon  de  panse),  473 

Massowah  vibrio,  acquired  immunity 
against,  221;  action  of  specific  serum 
on,  305 

Mastzellen,  77 

Membranes,  protective  secretion  of,  by 
bacteria,  21,  242 

Meriones  shawii,  22,  183 

Mesenteric  glands,  62,  85,  98,  195 

Mesoderm,  function  of  amoeboid  cells  of, 
518 

Microbicidal.     See  Bactericidal 

Micrococcus  prodigiosus,  42,  45 ;  antagon- 
istic to  anthrax  bacillus,  323 ;  action  of 
vaginal  mucus  on,  430 

Microcytase  digests  bacteria,  196,  197,  296, 
550 ;  in  immunity,  218 ;  escape  of,  during 
phagolysis,  218,  222,  230,  295,  554; 
transforms  vibrios  into  granules,  552 ; 
action  of,  on  Vibrio  metchnikovi,  553 

Micro-organisms,  minuteness  of  certain 
pathogenic,  3 ;  variability  in  action  of, 
5 ;  staining  reactions  of,  13,  83,  181, 
183,  198,  213 ;  immunity  by  attenuated, 
2,  509;  pathogenic,  in  healthy  persons, 
7 ;  adaptation  of,  to  toxic  substances, 
21,  25 ;  protective  secretion  of  mem- 
branes by,  21,  242;  defence  in  plants 
against,  35 ;  defences  of  animals  against, 
545 ;  elimination  of,  from  the  body,  43, 
46  (see  also  Elimination) ;  resorption 
of,  46,  175,  546;  antidiastase  against 
enzymes  of,  109 ;  natural  immunity 
against  pathogenic,  128-174,  175-206; 
acquired  immunity  against  pathogenic, 
207-249,  250-299,  300-324;  anaerobic, 
immunity  against,  169,  170 ;  pathogenic 
animal,  2,  173,  247-249,  277-279,  316; 
destruction  of,  an  act  of  resorption, 
175,  206  (see  Bacteriolysis) ;  presence  of, 
in  white  corpuscles,  514 ;  adaptation  of 
phagocytes  to  destroy,  558,  566 ;  mode 
of  entry  into  phagocytes,  177 ;  digested 
by  phagocytes,  181,  514-525,  536,  539- 
543  (see  Phagocytes,  Phagocytosis) ;  trans- 
formation into  spherical  granules,  198 
(see  also  Pfeiffer's  phenomenon) ;  extra- 
cellular destruction  of,  165,  212,  533- 
537,  542;  modified  growth  in  serums  from 
immunised  animals,  256,  259  (see  also 
Agglutination) ;  specific  diagnosis  of,  by 
modified  growth,  256,  259 ;  agglutination 
does  not  prevent  growth  of,  262 ;  changes 
which  they  undergo  in  immunised  animal, 
289  ;  attenuation  of,  '208,  286-289,  508 ; 
adjuvant  and  retarding  functions  of, 
170,  426 ;  antagonism  between  anthrax 
and  certain,  323 ;  antagonism  between 
cholera  vibrio  and  certain,  324;  acido- 
phile,  418;  exfoliation  of  epidermis  to 
get  rid  of,  406;  localisation  and  arrest 


Index 


585 


of,  in  the  dermis,  406;  destruction  of 
toxins  by,  427 

Microphages,  77,  78,  79,  148,  152,  154, 
162,  164,  172,  185,  245,  548;  intervene 
specially  against  micro-organisms  and 
in  acute  infections,  177,  196,  206,  549; 
source  of  bactericidal  substance  in  body 
fluids,  187,  195;  granular  transforma- 
tion of  vibrios  inside,  164,  165,  224  (see 
also  Pfeiffer's  phenomenon) 

Microsphaera,  18 

Milk,  absorption  of,  107  ;  precipitins  in  the 
differentiation  of  various  kinds  of,  107, 568 ; 
of  immunised  animals,  antitoxin  in,  356 ; 
immunity  conferred  by  mother's,  449, 
450,  452 ;  transmission  of  agglutinative 
power  by,  450 

Milk-sugar,  adaptation  of  yeasts  to,  26 

Mithridates,  method  of  protecting  himself 
against  poisons,  343 

MoUusca.  See  also  Helix,  Phyllirhoe,  Thetys 

Mollusca,  natural  immunity  in,  134 ;  liver 
of,  an  organ  of  second  digestion,  59 

Mongoose,  immunity  of,  against  snake 
venom,  339 

Monkeys,  immunised,  with  poor  yield  in 
antitoxin,  373  ;  immunisation  of,  against 
diphtheria  toxin,  373  ;  transient  acquired 
immunity  against  recurrent  fever,  434 

Monospora,  parasite  of  Daphnia  disease, 
131,  404,  520 

Morphia,  adaptation  to,  343 

Mouse,  infection  of,  by  swine  erysipelas, 
270,  307,  476 ;  the  only  animal  that 
acquires  immunity  by  suckling,  450,  452  ; 
acquired  immunity  of,  against  typhoid, 
230  ;  natural  immunity  of,  against  diph- 
theria toxin,  204,  339 

Mouse  septicaemia,  immunity  of  frog 
against,  141 ;  phagocytosis  in,  283 ;  ac- 
quired immunity  of  rabbit  against,  509 

Mouth.     See  Buccal  cavity 

Mucous  membranes,  immunity  of  the,  407- 
432 ;  elimination  of  micro-organisms 
by  the  nasal,  410 ;  protective  function  of 
the  genital,  429 

Mycoses,  pulmonary,  413  (see  also  Asper- 
gillosis) 

My  gale.    See  Spiders 

Myriapods.     See  Scolopendra 

Myxomycetes,  plasmodia  of,  30,  545 

Naegeli's  theory  of  immunity,  512 
Nagana  disease,  2,  4,  247,  316  (see  Trypano- 

soma) 

Narcosis.     See  Opium 
Nasal  mucous  membrane,   elimination  of 

organisms  by,  410 
Nepenthes,  digestive  juice  of,  355 
Nerve  centres,  susceptibility  of,  to  toxins,  564 
Neuroglia  cells,  their  phagocytic  function,  75 
Neurotoxin,  116 
Neutral  red,  reaction  of,  13,  83,  181 


Nuclein   as  a  protective    substance,   320 ; 

vaccinal  against  plague,  490 
Nucleohiston,    preventive    action    of,    on 

diphtheria  toxin,  365 
Nutrition,  certain  diseases  of,  probably  due 

to  a  parasite,  3  ;  extrabuccal,  67,  69 

Oidium  albicans,  growth  of,  in  serum  of 
immunised  animals,  257 

Omentum,  glands  of,  85 ;  bactericidal 
power  of  extracts  of,  195 ;  phagocytosis 
of  vibrios  in,  224 

Opium,  its  action  on  leucocytes,  225,  231, 
236,  306,  307;  its  influence  on  immu- 
nisation by  specific  serums,  306 ;  resist- 
ance of  hedgehog  to,  337 

Oryctes  nasicornis.    See  Ehinoceros  beetle 

Osmotic  pressure,  adaptation  of  plants  to, 
37,  39,  566;  as  cause  of  bactericidal 
action  of  body  fluids,  193,  213 

Ovum  in  the  Graafian  follicle,  immunity 
acquired  by  the,  448 

Oxalic  acid,  function  of,  in  plants,  37,  566 

Oxydases,  96 

Pancreatic  digestion,  60,  63,  65 

Pancreatic  juice,  antitoxic  power  of,  424 

Pancreatic  secretion,  its  adaptation  to  kind 
of  food,  64,  65 

Paralysis,  general  progressive,  and  syphilis, 
435 

Paramaecia,  13,  16,  17,  19 

Parasites  in  infective  diseases,  2,  9  (see 
also  Micro-organisms) 

Pasteur's  theory  of  exhaustion  of  nutrient 
medium,  510-512  ;  anthrax  vaccines,  208, 
470;  modification  of  Willems'  method 
against  pleuropneumonia,  477 ;  vaccines 
against  rabies,  462,  463-464 ;  and  Thuil- 
lier's  vaccines  against  swine  erysipelas, 
208,  473,  509 

Pepsin  in  the  urine,  65,  97 ;  in  the  blood, 
66,  563 ;  antitoxic  function  of,  419 ;  anti- 
septic action  of,  417 ;  chemical  composition 
of,  199 

Pessimism  and  fear  of  disease,  1,  569 

Peyer's  patches,  61 ;  protective  function  of, 
428 

Peziza.    See  Sclerotinia 

Pfaundler's  reaction,  259 

Pfeiffer's  phenomenon  in  cholera  vibrio, 
165,  192,  212-226,  251,  267,  268,  280, 
301-307,  534—536;  in  spirillum  of  re- 
current fever,  229 ;  in  typhoid  bacillus, 
230,  303,  304 ;  in  Bacillus  pyocyaneus, 
234,  307 ;  different  hi  immunised  and  in 
normal  fluids,  251;  conditions  for  its 
manifestation,  252,  253,  295,  534 

Pfeiffer's  theory  of  immunity,  534 

Phagocytes  (see  also  Leucocytes),  amoeboid 
cells  with  digestive  function,  47,  182, 
547  ;  in  Sponges,  69  ;  in  Vertebrata,  73  ; 
various  categories  of,  74-79;  of  Bipin- 


586 


Index 


naria  and  Phyllirhoe',  70 ;  chemiotaxis  of, 
79, 108,  133,  167,  177,  280 ;  the  source  of 
the  haemolytic  ferment,  100 ;  of  osseous 
fishes,  135;  of  frog,  137;  ingest  living 
and  virulent  bacteria,  142,  177,  179-181, 
558,  566 ;  function  of,  151,  157,  177,  181, 
206, 547, 548, 566 ;  mode  of  entry  of  micro- 
organisms into,  177  ;  acid  reaction  inside, 
83,  182 ;  action  of  opium  on,  225,  231 ; 
theory  of,  and  side-chain  theory  com- 
pared, 296-299,  538;  in  defence  of  animal 
against  poisons,  393-400 ;  in  production 
of  antitoxin,  400-402 ;  in  the  defence  of 
the  skin,  407 ;  attempts  to  reconcile 
theory  of,  with  humoral  theory,  539; 
history  of  theory  of,  514-525,  539-543 ; 
stimulant  action  of,  532 

Phagocytic  crisis  of  Bordet,  314 ;  ferments, 
549-558 ;  function  of  neuroglia  cells,  75  ; 
organs,  85,  150,  292,  293,  537  ;  of  cricket, 
133 ;  of  Ascaris,  547 

Phagocytosis  in  osseous  fishes,  135 ;  in 
frogs,  137,  142 ;  in  fowl,  144,  282 ;  in 
dog,  149, 151 ;  in  rat,  154, 157  ;  in  guinea- 
pig,  162,  163,  166,  170,  223  ;  in  horse, 
245,  313 ;  in  rabbit,  159,  167,  169,  233, 
239,  314 ;  effect  of  removal  of  spleen  on, 
150;  agents  that  prevent,  170-173  (see 
also  Opium) ;  neutralisation  of  toxins  not 
necessary  for,  205,  289 ;  and  agglutina- 
tion, 202,  242,  245;  ensures  natural 
immunity,  206  ;  action  of  opium  on,  225, 
231,  236,  306,  307 ;  action  of  rabbit's 
serum  on,  231;  in  acquired  immunity, 
220,  223-226,  245,  280-286,  295,  313; 
relation  to  fixatives  in  acquired  immu- 
nity, 291,  295 ;  in  the  immunity  con- 
ferred by  specific  serums,  303-317; 
history  of,  and  of  the  theory  of  phago- 
cytes, 514-525,  539-543;  its  application 
in  surgery,  568 

Phagolysis,  80,  99,  165 ;  prevention  of,  99, 
218,  219,  220,  230,  252,  304 ;  its  relation 
to  extracellular  destruction  of  bacteria 
and  Pfeiffer's  phenomenon,  218-220,  230, 
280,  295,  534 ;  escape  of  cytases  during, 
95,  99,  102,  191,  197,  252,  551-554,  560 

Philocytase,  91,  92 

Phloridzin,  its  action  on  natural  immunity, 
150 

Phyllirhoe,  two  modes  of  digestion  in,  58 ; 
resorption  by  phagocytes  of,  70 

Pig.     See  also  Swine 

Pig,  protection  of,  against  tetanus,  493 

Pigeon,  immunity  of,  against  anthrax,  146 ; 
immunity  of,  against  human  tuberculosis, 
147;  immunity  of,  against  influenza 
bacillus,  130,  554 ;  its  blood  best  culture 
medium  for  influenza  bacillus,  130,  554 ; 
susceptible  to  swine  erysipelas,  476 ;  pro- 
tective power  of  serum  of,  immunised 
against  anthrax,  276,  277,  288;  vacci- 
nation of,  against  anthrax,  276,  277 


Pilocarpin  augments  production  of  anti- 
toxin, 380 

Piroplasma  bigeminum,  247,  279 

Plague,  bubonic,  rapid  immunisation  by 
serum,  312  ;  protective  influence  of  broth 
against,  321 ;  production  of  antitoxic 
serum  by,  401 ;  infection  by,  through 
the  nasal  cavity,  409,  411 ;  vaccinations 
against,  486-492;  serum  treatment  in, 
490-492 ;  immunity  against,  when  ac- 
quired and  duration,  488,  489 ;  statistics 
on  vaccinations  against,  488;  prophy- 
lactic treatment  against,  491 ;  Eeports  of 
German  and  English  Commissions  on,  489 

Planarians,  digestion  in,  49,  71,  82 

Plants,  immunity  in  multicellular,  29-39 ; 
cicatrisation  of,  34 ;  and  osmotic  pres- 
sure, 37,  39,  566 ;  ravages  of  Sclerotinia 
amongst  cultivated,  32;  action  of  ma- 
nures on  immunity  of  cultivated,  36; 
function  of  oxalic  acid  in,  37,  566 

Plasma,  Gengou's  method  of  preparing, 
157,  190 

Plasmas.    See  also  Body  fluids,  Serums 

Plasmas,  presence  of  fixatives  in,  103; 
bactericidal  power  of,  190,  543 

Plasmase  (fibrin  ferment),  95,  197,  550 

Plasmodia,  intracellular  digestion  in,  30, 
545 ;  chemiotaxis  of,  30 ;  adaptation  of, 
to  poisons,  30 

Pleuropneumonia,  bacterium  of,  3,  130,  478, 
569;  vaccinations  against,  477-479;  action 
of  serum  from  animals  immunised  against, 
479;  vaccinal  methods  used  by  savage 
races  against,  506 

Pneumococcus,  modified  growth  of,  in 
serums  fromimmunised  animals,  256, 262 ; 
vaccination  against,  262;  attenuated  by 
serums  from  vaccinated  animals,  287; 
agglutination  of,  287 

Pneumo-enteritis  of  swine,  cocco-bacillus 
of,  259;  action  of  serum  of  vaccinated 
rabbits  on  bacillus  of,  260,  266,  287,  532; 
acquired  immunity  against,  260,  275,  311, 
532 

Pneumonia,  fibrinous,  relapses  separated 
by  periods  of  immunity,  434 

Poisons.    See  also  Toxins 

Poisons,  absorption  of,  by  leucocytes,  393- 
400 

Polyphagus  euglenae,  12 

Potato  attacked  by  Bacillus  coli,  35 

Precipitins  in  the  blood  serum,  68, 106, 107 ; 
use  of,  in  medico-legal  investigations, 
107,  568;  and  in  the  differentiation  of 
various  kinds  of  milk,  107,  568 

Predisposition  or  absence  of  immunity,  7 

Preventive  substances  of  Bordet  (syn.  fixa- 
tives), 266 

Profetta,  law  of,  453 

Protective  or  anti-infective  property.  See 
also  Antitoxic,  Antitoxins,  Blood,  Body 
fluids,  Serums 


Index 


587 


Protective  property,  origin  of,  in  serums 
and  other  fluids,  291-294;  differs  from 
agglutinative,  268,  269,  294;  of  blood  and 
other  fluids  in  convalescents,  437-444 

Protective  action  of  normal  serums,  317- 
320 ;  of  fats  against  toxins,  387 ;  of  leu- 
cocytes against  poisons,  393-400;  of  flow 
of  a  fluid,  431 

Protective  function  of  the  skin,  404-407 ;  in 
the  respiratory  channels,  411-414 ;  of  the 
cornea,  409 ;  of  the  saliva,  415 ;  of  the 
intestine,  422 ;  of  the  bile,  424 ;  of  the 
liver,  427 ;  of  the  lymphoid  organs,  428 ; 
of  the  suprarenal  capsules,  431;  in  the 
urinary  organs,  431 

Protective  substance  resistant  to  heat,  268 ; 
and  so  distinguished  from  bactericidal 
substance,  268;  closely  connected  with 
fixative  substance,  269,  294,  295,  561 

Protective  vaccinations,  454-504 

Proteus  vulgaris,  susceptibility  of  leucocytes 
to,  166,  179,  201,  282;  eosinophile  trans- 
formation in,  198;  modified  growth  in 
certain  serums,  259 

Protozoa,  intracellular  digestion  in  the,  13, 
30,  49 ;  adaptation  of,  to  saline  solutions, 
23,  515 ;  and  to  physical  conditions,  26 

Prussia  acid,  antidote  to,  363 

Pseudo-diphtheria  bacilli,  444 

Pseudo-eosinophile  leucocytes,  secretion  by, 
187,  542 

Pseudo-immunity  or  resistance,  320 

Pus,  ferment  in,  96 

Pyrogallic  acid,  its  action  on  natural  im- 
munity, 150 

Rabbit,  immunity  of,  against  anthrax 
bacillus,  159;  against  streptococci,  167, 
168;  against  tetanus  bacillus,  169 ;  against 
cholera  vibrio,  424;  against  pleuropneu- 
monia,  569 ;  acquired  immunity  of, 
against  pyocyanic  disease,  232;  against 
swine  erysipelas,  236-239,  527;  against 
anthrax,  239,  323;  against  streptococcus, 
243-247,  284-286,  312,  314;  against 
pneumo-enteritis,  260,  266,  275,  311, 
532 ;  against  pneumococcus,  262 ;  against 
a  staphylococcus,  266;  against  hog  cho- 
lera, 290;  against  mouse  septicaemia, 
509;  phagocytosis  in,  159,  167,  169,  233, 
239, 314, 569;  infection  by  streptococci  in, 
283;  action  of  serum  of  vaccinated,  on 
bacillus  of  pneumo-enteritis,  287 ;  action 
of  agglutinated  pneumococci  on,  287; 
vaccinated  against  hog  cholera  susceptible 
to  its  toxin,  290;  immunised  against  an- 
thrax by  means  of  the  erysipelas  coccus, 
323 ;  immunised  against  anthrax  by  pro- 
ducts of  Bacillus  pyocyaneus,  323;  in- 
fection by  anthrax  prevented  by  Fried- 
lander's  bacillus,  323;  brain  of,  very  sus- 
ceptible to  action  of  tetanus  toxin,  383 ; 
reaction  of,  to  atropin,  395 


Babies,  action  of  anthrax  bacillus  on,  150; 
action  of  normal  ox  serum  on,  365 ;  action 
of  bile  on,  425;  heredity  of  immunity 
against,  446 ;  vaccinations  against,  461- 
466;  statistics  of  vaccinations  against, 
464-466;  in  domestic  animals,  vaccina- 
tions against,  466 

Rat,  immunity  of,  against  anthrax  bacillus, 
152,  526;  against  diphtheria  bacillus, 
204 ;  acquired  immunity  against  Trypano- 
somata,  247-249,  316;  against  anthrax, 
240;  natural  immunity  of,  against  diph- 
theria toxin,  204,  339;  bactericidal  fer- 
ment of  phagocytes  of,  20,  157;  phago- 
cytosis in,  154,  157 

Receptors,  93, 120,  296;  over-production  of, 
121,  296,  562;  antitoxic  and  philotoxic 
functions  of,  120;  theory  of,  see  Side- 
chain  theory 

Recurrent  fever.  See  Spirilla,  Spirochaete 
obermeyeri 

Recurrent  fever,  transient  acquired  im- 
munity against,  434 

Rennet,  109,  119 

Reptilia.  See  Alligator,  Turtle,  Snake, 
Lizard 

Reptilia,  natural  immunity  of,  against  te- 
tanus toxin,  331-334 

Resistance  to  disease,  8-10.  See  Immunity, 
Pseudo-immunity 

Resorption  of  micro-organisms,  46,  175  (see 
also  Immunity,cellular,Micro-organisms); 
of  the  formed  elements,  47, 67-105;  a  true 
intracellular  digestion,  85,  296;  of  cells 
in  the  Invertebrata,  70 ;  of  red  corpuscles 
by  phagocytes  of  the  Vertebrata,  72,  80 
(see  also  Phagocytes,  Phagocytosis) ;  part 
played  by  macrophages  in  (see  Macro- 
phages) ;  and  digestion  closely  related, 
69,  85;  of  spermatozoa,  84,  100;  of 
white  corpuscles,  84  (see  also  Leucocytes, 
Phagocytes);  of  albuminoid  substances, 
106-127 ;  of  cells  and  the  phenomena  in 
acquired  immunity,  296 

Respiratory  channels,  protection  by  the, 
411-414;  absorption  of  poisons  by  the, 
414 

Rhinoceros  beetle,  natural  immunity  in 
larvae  of,  132,  209,  326,  329;  suscepti- 
bility to  cholera  vibrio,  40,  133 

Ricin,  344,  360,  446,  449 

Rinderpest,  action  of  bile  on,  425,  466; 
vaccinations  against,  466-468;  Koch's 
method  of  vaccination  against,  466; 
Kolle  and  Turner's  method  of  "simul- 
taneous vaccinations"  against,  467 

Ring-worm,  mechanical  factor  in,  4 

Robin  (toxalbumin  of  Eobinia  pseudacacia), 

.  365 ;  serum  of  animals  vaccinated  against, 
antitoxic,  365;  heredity  of  immunity 
against,  446 

Saccharomyces.    See  Yeasts 


588 


Index 


Saline  solution  (physiological)  as  a  protec- 
tive fluid,  320,  365 

Saliva,  microbicidal  property  of  the,  415; 
antitoxic  function  of,  on  snake  venom, 
417  ;  psychic  influence  on  flow  of,  62,  566 

Saponin,  haemolytic  action  of,  389;  and 
cholesterin,  389;  and  antisaponic  power, 
390 

Saprolegnia.     See  Fungi 

Sarcinae  as  adjuvant  organisms,  426 

Sarcinae,  acidophile,  418 

Sclerotinia,  pathogenic  action  of,  32 

Scolopendra,  acquired  immunity  in,  against 
anthrax,  209 

Scorpion,  natural  immunity  of,  against 
tetanus  toxin,  326;  against  its  own 
poison,  327;  antivenomous  property  of 
blood  of,  328;  supposed  suicide  of,  327 

Scorpion  serum,  action  of  antivenomous 
serum  on,  365 

Scorpion  venom,  antitoxic  action  of  cray- 
fish blood  against,  366 

Scrofula  in  immunity  against  tuberculosis, 
436 

Secretion  of  bactericidal  substance,  theory 
of,  187-191,  533-537,  540,  542 

Sensibilising  substance  of  Bordet  (fixative), 
91,  199,  298,  535,  537,  557 

Sensitiveness  of  plants  to  osmotic  pres- 
sure, 37,  566 

Septicaemia  of  goose.  See  Spirochaete  an- 
serina 

Septicaemia  of  mouse.  See  Mouse  septi- 
caemia 

Septic  vibrio,  170 

Serums.  See  also  Blood,  Body  fluids, 
Humoral  theory,  Toxins 

Serums,  haemolysis  by,  83,  87-95  (see  also 
Haemolysis);  effect  of  injections  of,  68; 
increasing  haemolytic  power  of,  90;  iso- 
toxic,  104;  absorption  of,  106  ;  antihae- 
motoxic,  111,  112;  haemolytic  or  hae- 
motoxic,  111,  112;  anticoagulating,  190; 
anticytase,  115,  371;  antispermotoxic, 
116,  122-126;  bactericidal  properties  of, 
184,  190,  191, 192, 193,  206, 211, 226,  233, 
238, 241,  243, 244, 260,  298,  554;  influence 
of  alkalinity  or  acidity  on  bactericidal 
action  of,  196;  agglutination  of  red  blood 
corpuscles  by,  258;  agglutination  of  bac- 
teria by,  256-265,  380;  protective  power 
of,  in  the  immunised  organism,  266-280, 
287,  293,  295,  532 ;  differs  from  bacteri- 
cidal power,  268 ;  and  from  agglutinative 
power,  268;  and  is  not  a  measure  of 
acquired  immunity,  271,  274,  275 ;  pro- 
tective, may  be  only  feebly  antitoxic, 
497;  modified  growth  of  bacteria  in  im- 
munised, 256,  259  (see  also  Agglutina- 
tion) ;  resistance  to  heat  of  protective 
substance  of,  268 ;  fixatives  in  protective, 
269,  438;  their  origin,  294;  protective 
and  fixative  substances  contrasted,  269; 


relations  of  fixative  and  cytase  in  bac- 
tericidal action  of,  298 ;  stimulating  action 
of,  270-274,  301,  308-320,  365 ;  absence 
of  protective  power  in  specific,  270,  276- 
279;  origin  of  protective  power  in,  291- 
294;  theory  of  attenuation  of  micro- 
organisms by  immune,  286-289 ;  inactive 
specific,  rendered  active  by  addition  of 
normal  serum,  215,  268,  298,  302,  317; 
protective  action  of  heated  normal  serum, 
273,  318;  protective  action  of  non-spe- 
cific, against  toxins,  365;  from  con- 
valescents, protective  action  of,  437-444 ; 
temporary  immunity  against  micro-or- 
ganisms conferred  by  specific,  301-317; 
conferred  by  normal,  317-320;  conferred 
by  fluids  other  than,  320-322 ;  phagocy- 
tosis in  the  immunity  conferred  by  spe- 
cific, 303-306;  influence  of  opium  on 
immunisation  by  specific,  306;  antive- 
nomous action  of,  334, 338,  358,  360,  361 ; 
antitoxic  action  of  non-specific  and  nor- 
mal, 365,  380;  anti-arsenic,  390;  anti- 
leucocidic,  359;  antidiastatic,  361 ;  testing 
and  standardisation  of  antitoxic,  376, 
476,  496-498 

Sheath,  protective.     See  Membrane 

Sheep,  natural  immunity  of,  against  an- 
thrax, 159,  289 ;  acquired  immunity  of, 
against  anthrax,  241-3,  289 ;  bactericidal 
action  of  blood  serum  of,  241,  286;  pro- 
tective power  of  serum  of,  immunised 
against  anthrax,  276;  immunised  with 
blood  from  dog  affected  by  a  haematozoon, 
279;  vaccination  of,  against  sheep-pox, 
460 ;  against  rabies,  466 ;  against  anthrax, 
469 ;  protection  against  tetanus  in,  493 ; 
fate  of  anthrax  bacilli  in  Algerian,  512 

Sheep-pox  (la  clavele"e),  heredity  of  immunity 
against,  452 ;  vaccinations  against,  460- 
461 

Side-chains  or  receptors,  theory  of,  120, 
381-384,  538,  557,  562-563;  compared 
with  theory  of  phagocytes,  296-299,  538, 
558 

Silver,  soluble  salts  of,  absorbed  by  leu- 
cocytes, 400 

Skin,  immunity  of  the,  403-407;  protective 
function  of  the,  404-407 ;  phagocytes  in 
the  defence  of  the,  407 

Small-pox,  mortality  from,  in  18th  century, 
454;  vaccinations  against,  454-460;  vac- 
cination with  calf  lymph,  456;  with  con- 
tents of  pustule  of  cow-pox,  455;  vac- 
cination statistics,  457-459 

Snail.     See  Helix  pomatia 

Snake,  natural  immuni'ty  of,  against  snake 
venom,  333 

Snake  venom,  natural  immunity  of  snakes 
against,  333;  of  hedgehog  against,  337; 
of  mongoose  against,  339 ;  artificial  im- 
munity against,  345,  347 ;  action  of  anti- 
venomous  serum  on,  358,  360,  361;  of 


Index 


589 


other  specific  serums  on,  365;  of  cerebral 
substance  on,  386;  protective  substances 
against,  387;  action  of  saliva  on,  417; 
action  of  bile  on,  425 ;  vaccination  methods 
of  savage  races  against,  506 

Spermatozoa,  resorption  of,  84,  100;  action 
of  spermotoxin  on,  101,  116,  125 

Spermotoxin,  101,  116,  125 

Spiders,  natural  immunity  of,  against 
tetanus  toxin,  326 

Spirilla,  natural  immunity  against,  159; 
acquired  immunity  against,  227-230,  434 ; 
living  in  stomach  of  dog,  177;  acidophile, 
418 

Spirochaete  anserina,  160 

Spirochaete  obermeyeri,  160;  acquired  im- 
munity against,  227-230;  Pfeiffer's  phe- 
nomenon in,  229 

Spleen,  function  of,  62,  85;  action  of 
extract  of,  on  tetanus  toxin,  365;  effect 
of  removal  of,  150,  293;  as  source  of 
fixative  substance,  295,  537 

Spleen  and  other  haematopoietic  organs  as 
source  of  protective  substance,  292-294; 
as  source  of  agglutinins,  264;  are  phago- 
cytic  organs,  85,  150,  292 

Sponges,  digestion  of,  69,  517 

Staining  reactions  of  cells  and  micro-or- 
ganisms, 13,  77,  83,  181,  183,  198,  213 

Standardisation  of  antidiphtheria  serums, 
376,  496-498;  Ehrlich's  method,  496; 
Pasteur  Institute  method,  496-497 

Staphylococcus,  acquired  immunity  against, 
266,'  532;  protective  action  of  normal 
serum  against,  319 

Staphylococcus  pyogenes  in  vagina,  430 

Stellate  cells  of  Kupffer,  75 

Stimulant  action.  See  also  Body  fluids, 
Protective 

Stimulant  action  of  serums,  270-274,  301, 
308-320,  365;  of  phagocytes,  532;  of 
normal  fluids  of  the  body,  559 

Stimulins  and  their  action  in  serums,  270- 
274 

Stohr's  phenomenon,  429 

Stomach,  acidophile  microbian  flora  of, 
418 

Streptococci,  protective  sheath  formed  by, 
22;  immunity  against,  165,  179,  282, 
284-286;  phagocytosis  in  immunity 
against,  245,  313;  acquired  immunity 
against,  243-247,  313;  agglutination  by 
serum  of,  244,  245;  reaction  of  animal 
organism  against,  245-247;  antitoxin 
against,  205;  and  phagocytosis,  283; 
action  of  specific  serums  on,  287,  288, 
312;  protective  action  of  various  fluids 
against,  320,  321 

Streptococcic  serum,  action  of,  on  leuco- 
cidin,  359 

Sturin,  bactericidal  action  of,  183 

Suprarenal  capsules,  protective  function 
of,  431 


Susceptibility.  See  also  Chemiotaxis,  Hyper- 
susceptibility,  Irritability,  Sensitiveness 

Susceptibility  of  immunised  animals  to 
the  specific  toxin,  289;  of  frogs  to  tetanus 
toxin,  330  ;  diminution  of,  in  immunised 
animals,  374-376 ;  in  immunity,  the  part 
played  by,  565;  cellular,  a  general  pro- 
perty of  living  beings,  565-566 

Swine.     See  Pig,  Pneumo-enteritis 

Swine  erysipelas,  acquired  immunity  against, 
236-239,  254,  283,  527 ;  agglutination  of 
bacilli  of,  262 ;  specific  serum  of,  will 
not  prevent  infection,  270;  phagocytosis 
in,  283;  action  of  immune  serums  on 
bacillus  of,  288,  289 ;  protective  action  of 
specific  serum  against,  307;  method  of 
testing  strength  of  serums  against,  476; 
vaccinations  against,  473-477;  Pasteur's 
method,  473;  Lorenz's  method,  475; 
"serum -vaccinations"  method,  475;  vac- 
cines against,  208,  473,  509 

Swine  plague,  259,  260 

Synapta,  518 

Syphilis,  immunity  in,  435;  and  general 
progressive  paralysis,  435;  law  of  Pro- 
fetta  in  immunity  against,  453;  law  of 
Baumes-Colles  in,  436;  transmission  of, 
452 

Tears,  microbicidal  function  of  the,  408 

Testing  of  serums.     See  Standardisation 

Tetanolysin  of  Ehrlich,  349 

Tetanospasmin,  362 

Tetanus,  immunisation  against,  344,  347, 
492-495;  cerebral,  in  rabbit,  383,  391; 
difference  between  antitoxic  action  of 
living  brain  and  that  of  cerebral  emul- 
sion on,  383 ;  in  fowl,  384 ;  no  antitetanic 
power  in  serum  of  convalescents,  443; 
vaccinations  against,  492-495;  vaccines 
against,  493 ;  protective  serum  treatment 
against,  493-495 

Tetanus  antitoxin,  hypothesis  of  nervous 
origin  of,  381-385 ;  nature  of,  355 ;  mode 
of  action  on  toxin,  357,  381;  of  nerve 
centres  locally  restricted  in  its  action,  382 

Tetanus  bacillus,  natural  immunity  against, 
169,  204 

Tetanus  toxin,  natural  immunity  of  spiders 
and  scorpions  against,  326 ;  of  larvae  of 
Oryctes  and  of  cricket  against,  329;  of 
frog  against,  330;  of  reptiles  against, 
331-334;  of  fowl  against,  335;  of  hiber- 
nating animals  against,  339;  attenuation 
of,  344 ;  localisation  of,  in  vascular  organs, 
336;  brain  of  rabbit  very  susceptible  to 
action  of,  383;  fixation  of,  by  substance 
of  nerve  centres,  382;  by  certain  parts  of 
brain  and  cord,  386,  391 ;  by  other  cells, 
391,  392;  action  of  emulsions  of  frog's 
brain  on,  386;  fixation  of,  by  carmine, 
388,  394;  absorption  of,  by  leucocytes, 
393-395;  action  of  extract  of  spleen  on, 


590 


Index 


365;  toxone  (tetanolysin)  of,  349,  362; 
local  reaction  to,  in  horse,  352 ;  heredity 
of  immunity  against,  446,  448,  450 

Texas  fever,  acquired  immunity  of  Bovidae 
against,  247,  279 ;  attenuation  of  parasite 
of,  in  the  tick,  247 ;  haematozoon  in  dog 
closely  allied  to  that  of,  279 

Thetys,  517 

Thymus  gland,  immunising  power  of,  293 

Tick,  attenuation  of  parasite  of  Texas  fever 
in,  247 

Tonsils,  protective  function  of,  428 

Torulae  as  adjuvant  organisms,  426 

Toxins,  immunity  against,  10;  immunity 
of  unicellular  organisms  against,  19; 
adaptation  of  bacteria  to,  21-27;  of  yeasts 
to,  20,  26;  of  plasmodiato,  30;  action  of, 
on  Infusoria,  19,  326;  composition  of, 
120;  neutralisation  of,  not  necessary  for 
phagocytosis,  205,  289;  immunity  against 
micro-organism  not  same  as  against 
toxin,  251,  290 ;  protective  fixation  of,  by 
nerve  elements  and  other  cells,  386-400 ; 
methods  of  immunisation  by  modified 
and  unmodified,  345-347  (see  Immunisa- 
tion); local  reaction  in  immunisation 
against,  352;  action  of  normal  serums 
on,  365 ;  of  non-specific  serums  on,  365 ; 
protective  action  of  fats  against,  387; 
leucocytic  reaction  against,  393-400; 
absorption  of,  by  the  conjunctiva,  409; 
by  the  respiratory  channels,  414;  de- 
struction of,  by  the  intestinal  organisms, 
427;  attenuation  of,  344;  natural  im- 
munity against,  325-341;  artificial  im- 
munity against,  342-402;  against  bac- 
terial, 343;  against  vegetable,  344,  365; 
heredity  of  immunity  in  phanerogamic, 
446,  449  j  susceptibility  of  nerve  centres 
to,  564 

Toxoids,  349  (see  also  Toxophore);  im- 
munisation by,  350 

Toxones,  349,  362;  method  of  immunisa- 
tion by,  349 

Toxophore  atomic  group  in  toxin  ( = toxoid) , 
120,  350,  384 

Trichinae,  mechanical  action  of,  3 

Tristeza  (syn.  Texas  fever),  247 

Tropidonotus.     See  Snake 

Trypanosoma,  4,  129,  147;  brucei,  9; 
lewisi,  173,  248 

Trypanosomata^i&te  of,  in  refractory  animal, 
173;  acquired  immunity  against,  247- 
249,  316;  and  agglutinative  power,  278 

Trypsin,  antitoxic  power  of,  424 

Tsetse  fly,  4,  9,  129,  247 

Tubercle  bacillus,  formation  of  sheath  by. 
22,  183 

Tuberculin  as  a  protective  substance  against 
cholera  vibrio,  320 

Tuberculosis,  mechanical  etiological  factors 
in,  4 

Tuberculosis,  bacillus  of,  22, 42, 143;  avian, 


41,  148,  149,  182 ;  human,  immunity  of 
pigeon  against,  147;  acquired  immunity 
in,  436;  after  scrofula,  436;  Koch's 
phenomenon  in,  437 

Tumours,  malignant,  probability  of  dis- 
covery of  parasite  of,  3 ;  use  of  erysipelas 
streptococcus  in,  434 

Turtle,  natural  immunity  of,  against  tetanus 
toxin,  332,  386 

Typhoid,  protective  power  of  serum  of  con- 
valescents from,  437-440;  its  agglutina- 
tive power,  439;  serum  diagnosis  of,  256, 
257,  261,  439;  immunity  against,  not 
acquired  by  suckling,  450;  vaccinations 
against,  479,  481-486 ;  Wright's  vaccine 
against,  482 ;  bactericidal  power  of  serum 
from  persons  immunised  against,  483; 
statistics  of  vaccinations  against,  483- 
485 

Typhoid  bacillus,  23,  143,  191,  198,  203; 
acquired  immunity  against,  230;  at- 
tenuated Pfeiffer's  phenomenon  in,  230, 
303,  304;  agglutination  of,  260,  261, 
380,  439 ;  resistance  to  agglutinated,  263 ; 
protective  action  of  serums  against,  272- 
274,  293,  317,  319;  origin  of  protective 
substance  against,  292;  of  agglutinative 
property  against,  294 ;  protective  action 
of  various  fluids  against,  320 ;  passes  un- 
injured through  stomach,  418;  trans- 
mission by  suckling,  of  agglutinative 
power  against,  450 

Typhoid  infection,  experimental,  in  labora- 
tory animals,  230,  267;  influence  of 
anticytase  serum  on,  371;  uncertainty  of, 
by  ingestion,  423 

Typhoid  septicaemia,  experimental,  here- 
dity of  immunity  against,  447 

Tyrosin,  protective  action  of,  387 

Unicellular  organisms,  immunity  in,  11-28; 

infective  diseases  of,  12;  irritability  of, 

27;  adaptation  of,  to  saline  solutions,  23, 

515 

Unit,  Ehrlich's  immunising,  373,  496 
Urinary  ferments,  66 
Urinary  organs,  protective  function  in,  431 
Urine  as  a  protective  fluid,  320,  431 ;  pepsin 

in  the,  65 ;  amylase  in  the,  65 

Vaccination.     See  also  Immunisation 
Vaccinations,    protective,    208,    241,    267, 
454-504,   507;   with  attenuated   micro- 
organisms, 509 

Vaccine  against  fowl  cholera,  208 
Vaccines  against  anthrax,  208,  470,  509; 
against  swine  erysipelas,  208,  473,  509; 
against  rabies,  208,  462,  463-464;  against 
symptomatic  anthrax,  471 ;  against  small- 
pox, 455-457,  507;  against  pleuropneu- 
monia,  477;  against  cholera,  481;  against 
plague,  487,  489,  490;  against  tetanus, 
493 


Index 


591 


Vaccinia,  supposed  micro-organism  of,  455- 

456 

Vagina,  autopurification  of,  429 
Variolisation,  early  use  of,  455,  507 
Venom.     See  Snake  venom 
Ver  blanc,  syn.  cockchafer  larva 
Vibrio.     See  also  Cholera  vibrio,  Massowah 

vibrio,  Septic  vibrio 

Vibrios,  acquired  immunity  against,  211- 
227 ;  phagocytosis  in  immunity  against, 
-  220, 223-226;  granular  transformation  of, 
164,  165,  192,  212-226  (see  Pfeiffer's 
phenomenon) ;  bacteriolysis  (agglutina- 
tion) of,  256;  susceptibility  of  animals 
vaccinated  against,  to  the  toxins,  290 
Vibrio  metchnikovi,  acquired  immunity 
against,  211,  226,  527,  531;  modified 
growth  of,  in  serums  from  immunised 
animals,  156,  262;  action  of,  grown  in 
serum  of  vaccinated  animals,  287;  perishes 
in  intestine  of  dog,  422 ;  action  of  micro- 
cytase  on,  in  hypervaccinated  guinea- 
pigs,  553 

Viper.     See  Snake,  Snake  venom 
Viruses,  attenuated,  208,  508;  vaccination 
with,  whose  nature  is  as  yet  unknown. 


See  Small-pox,  Sheep-pox,  Babies,  Rin- 
derpest 

Vitellus  of  egg  of  immunised  fowl,  tetanus 
antitoxin  present  in,  356 ;  immunity  con- 
ferred by,  449 

Warlomont's  calf  lymph  vaccine,  456 
Weber-Fechner,  law  of,  27,  38,  566 
Willem's  method    of   vaccination    against 
pleuropneumonia,  477 ;  Pasteur's  modifi- 
cation of,  477 

Wright's  method  of  vaccination  against 
typhoid,  482;  method  of  testing  bacteri- 
cidal power  of  body  fluids,  483 

Yeast-cells,  adaptation  of,  to  poisons,  20, 
26;  to  milk-sugar,  26;  destruction  of 
injected,  by  phagocytes,  172;  Curtis's 
pathogenic,  172;  endotrypsin  of,  197; 
autodigestion  in,  197;  soluble  ferments 
of,  253 

Yeasts,  diseases  due  to,  2 

Yolk.    See  Vitellus 

Zymase,  197,  550 


CAMBRIDGE  :    PRINTED   BY   JOHN    CLAY,    M.A.   AT   THE   UNIVERSITY  PRESS. 


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